Activity-dependent mechanisms of axonal growth

Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut de Bioenginyeria de Catalunya (IBEC)Spinal cord injuries (SCI) are a major cause of paralysis in young adults. In this type of injuries recovery is impaired as adult central nervous system (CNS) axons fail to regenerate. This results from both a loss of intrinsic growing capacities in developmental axons when they mature, together with the presence of extrinsic factors hampering this regeneration, including a glial scar together with the production of growth-inhibitory molecules, as well as a lack of injury resolution leading to a chronic inflammation. Unfortunately, despite research efforts, current therapies for this type of injuries only lead to mild improvements and among them, activity-based therapies seem to raise above the others. Activity- based therapies try to induce recovery by increasing neuronal activity, however, a proper physiological and molecular characterization of the rationale behind their success is still missing. Neuronal activity has been described to regulate transcriptional and epigenetic mechanisms; moreover, it also alters neuronal secretion with an impact on cellular dialogues. These characteristics indicate neuronal activity may be modulating both of the CNS barriers for regeneration. During this doctoral thesis we aimed to explore the influence of neuronal activity on SCIs, hypothesizing specific neuronal activations were the principal responsible for success in activity-based therapies. Particularly, we studied the role of precise manipulations of neuronal activity, using optogenetic and chemogenetic tools, in axonal growth of stimulated neurons as well as the impact these activations could have on neuronal extrinsic signalling. Our results show that optogenetic and chemogenetic stimulations of neuronal activity enhanced growth in both regenerating and refractory to regenerate neurons. However, this growth was hampered by the inhibitory molecules present in the injured CNS and did not result in functional recovery in rodent models of SCI. Our data indicated that the growth induction in specifically stimulated neurons resulted from local adjustments rather than inducing a pro-regenerative transcriptional state, as seen by our gene expression analysis of regeneration-associated genes (RAGs). Altogether, our results suggest recovery in activity-based therapies derives from the summation of various forms of plasticity, induced by their simultaneous recruitment of several circuits. In parallel, we observed that these precise modulations of neuronal activity, while unable to alter the predominant environment after SCI, could initiate previously undescribed intricate cellular dialogues. Specifically, we found an increase in the chemokine CCL21 upon nociceptor activation which triggered the response of several cell types in the injury. In proprioceptors, this CCL21 was responsible for a growth induction after CCR7 activation, which required the MEK-ERK pathway as well as the modulation of the actin cytoskeleton. Meanwhile, the CCL21 interaction with CXCR3 in other cells effectively aborted this regeneration. All in all, our work reveals the existence of a complex plethora of synergic mechanisms, far from understood, contributing to the outcome of activity-based therapies and reinforces the need for further mechanistic studies which would allow the optimization of their success

Genetic control of neuronal activity enhances axonal growth only on permissive substrates

Abstract Background Neural tissue has limited regenerative ability. To cope with that, in recent years a diverse set of novel tools has been used to tailor neurostimulation therapies and promote functional regeneration after axonal injuries. Method In this report, we explore cell-specific methods to modulate neuronal activity, including opto- and chemogenetics to assess the effect of specific neuronal stimulation in the promotion of axonal regeneration after injury. Results Opto- and chemogenetic stimulations of neuronal activity elicited increased in vitro neurite outgrowth in both sensory and cortical neurons, as well as in vivo regeneration in the sciatic nerve, but not after spinal cord injury. Mechanistically, inhibitory substrates such as chondroitin sulfate proteoglycans block the activity induced increase in axonal growth. Conclusions We found that genetic modulations of neuronal activity on both dorsal root ganglia and corticospinal motor neurons increase their axonal growth capacity but only on permissive environments

Neuromuscular activity induces paracrine signaling and triggers axonal regrowth after injury in microfluidic lab‐on‐chip devices

Peripheral nerve injuries, including motor neuron axonal injury, often lead to functional impairments. Current therapies are mostly limited to surgical intervention after lesion, yet these interventions have limited success in restoring functionality. Current activity‐based therapies after axonal injuries are based on trial‐error approaches in which the details of the underlying cellular and molecular processes are largely unknown. Here we show the effects of the modulation of both neuronal and muscular activity with optogenetic approaches to assess the regenerative capacity of cultured motor neuron (MN) after lesion in a compartmentalized microfluidic‐assisted axotomy device. With increased neuronal activity, we observed an increase in the ratio of regrowing axons after injury in our peripheral‐injury model. Moreover, increasing muscular activity induces the liberation of leukemia inhibitory factor and glial cell line‐derived neurotrophic factor in a paracrine fashion that in turn triggers axonal regrowth of lesioned MN in our 3D hydrogel cultures. The relevance of our findings as well as the novel approaches used in this study could be useful not only after axotomy events but also in diseases affecting MN survival

Involvement of Mechanical Cues in the Migration of Cajal-Retzius Cells in the Marginal Zone During Neocortical Development

Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone

Activity-dependent mechanisms of axonal growth

[eng] Spinal cord injuries (SCI) are a major cause of paralysis in young adults. In this type of injuries recovery is impaired as adult central nervous system (CNS) axons fail to regenerate. This results from both a loss of intrinsic growing capacities in developmental axons when they mature, together with the presence of extrinsic factors hampering this regeneration, including a glial scar together with the production of growth-inhibitory molecules, as well as a lack of injury resolution leading to a chronic inflammation. Unfortunately, despite research efforts, current therapies for this type of injuries only lead to mild improvements and among them, activity-based therapies seem to raise above the others. Activity- based therapies try to induce recovery by increasing neuronal activity, however, a proper physiological and molecular characterization of the rationale behind their success is still missing. Neuronal activity has been described to regulate transcriptional and epigenetic mechanisms; moreover, it also alters neuronal secretion with an impact on cellular dialogues. These characteristics indicate neuronal activity may be modulating both of the CNS barriers for regeneration. During this doctoral thesis we aimed to explore the influence of neuronal activity on SCIs, hypothesizing specific neuronal activations were the principal responsible for success in activity-based therapies. Particularly, we studied the role of precise manipulations of neuronal activity, using optogenetic and chemogenetic tools, in axonal growth of stimulated neurons as well as the impact these activations could have on neuronal extrinsic signalling. Our results show that optogenetic and chemogenetic stimulations of neuronal activity enhanced growth in both regenerating and refractory to regenerate neurons. However, this growth was hampered by the inhibitory molecules present in the injured CNS and did not result in functional recovery in rodent models of SCI. Our data indicated that the growth induction in specifically stimulated neurons resulted from local adjustments rather than inducing a pro-regenerative transcriptional state, as seen by our gene expression analysis of regeneration-associated genes (RAGs). Altogether, our results suggest recovery in activity-based therapies derives from the summation of various forms of plasticity, induced by their simultaneous recruitment of several circuits. In parallel, we observed that these precise modulations of neuronal activity, while unable to alter the predominant environment after SCI, could initiate previously undescribed intricate cellular dialogues. Specifically, we found an increase in the chemokine CCL21 upon nociceptor activation which triggered the response of several cell types in the injury. In proprioceptors, this CCL21 was responsible for a growth induction after CCR7 activation, which required the MEK-ERK pathway as well as the modulation of the actin cytoskeleton. Meanwhile, the CCL21 interaction with CXCR3 in other cells effectively aborted this regeneration. All in all, our work reveals the existence of a complex plethora of synergic mechanisms, far from understood, contributing to the outcome of activity-based therapies and reinforces the need for further mechanistic studies which would allow the optimization of their success

Injury-induced activation of the endocannabinoid system promotes axon regeneration

Summary: Regeneration after a peripheral nerve injury still remains a challenge, due to the limited regenerative potential of axons after injury. While the endocannabinoid system (ECS) has been widely studied for its neuroprotective and analgesic effects, its role in axonal regeneration and during the conditioning lesion remains unexplored. In this study, we observed that a peripheral nerve injury induces axonal regeneration through an increase in the endocannabinoid tone. We also enhanced the regenerative capacity of dorsal root ganglia (DRG) neurons through the inhibition of endocannabinoid degradative enzyme MAGL or a CB1R agonist. Our results suggest that the ECS, via CB1R and PI3K-pAkt pathway activation, plays an important role in promoting the intrinsic regenerative capacity of sensory neurons after injury