Resumen de Boosting endogenous mechanisms for neuronal protection and repair after injury in central nervous system.
Neurons have endogenous neuroprotective mechanisms that are capable to activate against different insults that threaten their life. Spinal motoneurons (MNs) activate these mechanisms retrogradely when they suffer axotomy of the nerve. Those allow MNs to face perfectly surviving and even regenerate after distal axotomy (DA) of the nerve. However, when the injury is more proximal to their soma, such as in the case of nerve root traction or avulsion (root avulsion, RA), these mechanisms are deficiently activated leading to degeneration. We reasoned that boosting the endogenous mechanisms of neuroprotection naturally activated within MNs might be effective for neuroprotection. Thus, we first elucidate these mechanisms that MNs endogenously trigger after DA and then manage to get an agent capable to shift the molecular network that characterizes MN degeneration after RA into a neuroprotective one.
In order to accomplish our goal, we envisaged a strategy based on a systems biology approach. We chose unconventional drug discovery pathway to rely on systems and network-targeting more convey for complex diseases, instead of the classical single gene-protein- target tracking. In addition, drug repurposing presents several advantages in the translation from bench to bedside. The TPMS computational tool combines both considerations. The present work leading to a PhD thesis was initiated using TPMS to discover new neuroprotective therapies based on shifting the pro-death molecular network that MNs suffer after nerve root avulsion towards a neuroprotective and pro-regenerative profile similar to that triggered after DA.
Drug combinations that came up from TPMS analysis with the highest scores were further validated in vitro and in vivo in the first chapter. We scored them based on their neuroprotective and anti-inflammatory effect as well as the pro-regenerative profile the treatment engaged into MNs. The best combination was chosen and was termed NeuroHeal (NH). In addition, at the wet-lab, we further validated its molecular mechanism of action (MoA) previously predicted in silico by TPMS. Sirtuin 1 (SIRT1) was one of the key molecules that characterizes NH MoA, and we also demonstrated that it exerted neuroprotection when overexpressed by intrathecal delivery of a specific adeno-associated viral vector. We observed that NH activated the activity of SIRT1 and modulated the expression of other proteins like cytoskeletal motor proteins and integrinβ1 subunit. Moreover, oral administration of NH accelerated nerve regeneration and promoted functional recovery after sciatic nerve crush which was unexpected.
Regarding the second and third chapters, we confirmed that NH and its ability to activate SIRT1, had neuroprotective effects also in other species and models such as cranial MN death induced by hypoglossal nerve axotomy; and age-dependent model of spinal MN degeneration after nerve axotomy. Using the former, we verified that the activation of both SIRT1 and SIRT2 were necessary for axotomized cranial MN survival. Although SIRT2 inhibitors were claimed neuroprotectants to other neurodegenerative processes, it inhibition was detrimental in our model probably due to the increase of toxic pro-inflammatory cytokines. In the latest case, neonatal axotomized MNs that normally die by apoptosis could survive thanks to the controlled autophagy induced by NH treatment which revealed an unexpected mechanism of action.
With the aim to step forward to clinical translation, we generated a preclinical model of ventral nerve root avulsion and delayed reimplantation which better mimic the reality at the clinical practice. Chronic long-term NH treatment maintained the survival of MNs for six months, accelerated nerve regeneration, reduced denervated-induced muscular atrophy, and favored the formation of functional neuromuscular junctions.
Overall, these findings suggested that NH may be an effective pharmacological therapy to increase the survival of disconnected MNs and accelerate the recovery of motor function after peripheral nerve injuries.
