The Role of Metals in the Neuroregenerative Action of BDNF, GDNF, NGF and Other Neurotrophic Factors

Mature neurotrophic factors and their propeptides play key roles ranging from the regulation of neuronal growth and differentiation to prominent participation in neuronal survival and recovery after injury. Their signaling pathways sculpture neuronal circuits during brain development and regulate adaptive neuroplasticity. In addition, neurotrophic factors provide trophic support for damaged neurons, giving them a greater capacity to survive and maintain their potential to regenerate their axons. Therefore, the modulation of these factors can be a valuable target for treating or preventing neurologic disorders and age-dependent cognitive decline. Neuroregenerative medicine can take great advantage by the deepening of our knowledge on the molecular mechanisms underlying the properties of neurotrophic factors. It is indeed an intriguing topic that a significant interplay between neurotrophic factors and various metals can modulate the outcome of neuronal recovery. This review is particularly focused on the roles of GDNF, BDNF and NGF in motoneuron survival and recovery from injuries and evaluates the therapeutic potential of various neurotrophic factors in neuronal regeneration. The key role of metal homeostasis/dyshomeostasis and metal interaction with neurotrophic factors on neuronal pathophysiology is also highlighted as a novel mechanism and potential target for neuronal recovery. The progress in mechanistic studies in the field of neurotrophic factor-mediated neuroprotection and neural regeneration, aiming at a complete understanding of integrated pathways, offers possibilities for the development of novel neuroregenerative therapeutic approaches

Motoneuronal inflammasome activation triggers excessive neuroinflammation and impedes regeneration after sciatic nerve injury

Peripheral nerve injuries are accompanied by inflammatory reactions, over-activation of which may hinder recovery. Among pro-inflammatory pathways, inflammasomes are one of the most potent, leading to release of active IL-1β. Our aim was to understand how inflammasomes participate in central inflammatory reactions accompanying peripheral nerve injury.After axotomy of the sciatic nerve, priming and activation of the NLRP3 inflammasome was examined in cells of the spinal cord. Regeneration of the nerve was evaluated after coaptation using sciatic functional index measurements and retrograde tracing.In the first 3 days after the injury, elements of the NLRP3 inflammasome were markedly upregulated in the L4-L5 segments of the spinal cord, followed by assembly of the inflammasome and secretion of active IL-1β. Although glial cells are traditionally viewed as initiators of neuroinflammation, in this acute phase of inflammation, inflammasome activation was found exclusively in affected motoneurons of the ventral horn in our model. This process was significantly inhibited by 5-BDBD, a P2X4 receptor inhibitor and MCC950, a potent NLRP3 inhibitor. Although at later time points the NLRP3 protein was upregulated in microglia too, no signs of inflammasome activation were detected in these cells. Inhibition of inflammasome activation in motoneurons in the first days after nerve injury hindered development of microgliosis in the spinal cord. Moreover, P2X4 or inflammasome inhibition in the acute phase significantly enhanced nerve regeneration on both the morphological and the functional levels.Our results indicate that the central reaction initiated by sciatic nerve injury starts with inflammasome activation in motoneurons of the ventral horn, which triggers a complex inflammatory reaction and activation of microglia. Inhibition of neuronal inflammasome activation not only leads to a significant reduction of microgliosis, but has a beneficial effect on the recovery as well

Riluzole treatment modulates KCC2 and EAAT-2 receptor expression and Ca2+ accumulation following ventral root avulsion injury

Avulsion injury results in motoneuron death due to the increased excitotoxicity developing in the affected spinal segments. This study focused on possible short and long term molecular and receptor expression alterations which are thought to be linked to the excitotoxic events in the ventral horn with or without the anti-excitotoxic riluzole treatment. In our experimental model the left lumbar 4 and 5 (L4, 5) ventral roots of the spinal cord were avulsed. Treated animals received riluzole for 2 weeks. Riluzole is a compound that acts to block voltageactivated Na+ and Ca2+ channels. In control animals the L4, 5 ventral roots were avulsed without riluzole treatment. Expression of astrocytic EAAT-2 and that of KCC2 in motoneurons on the affected side of the L4 spinal segment were detected after the injury by confocal and dSTORM imaging, intracellular Ca2+ levels in motoneurons were quantified by electron microscopy. The KCC2 labeling in the lateral and ventrolateral parts of the L4 ventral horn was weaker compared with the medial part of L4 ventral horn in both groups. Riluzole treatment dramatically enhanced motoneuron survival but was not able to prevent the down-regulation of KCC2 expression in injured motoneurons. In contrast, riluzole successfully obviated the increase of intracellular calcium level and the decrease of EAAT-2 expression in astrocytes compared with untreated injured animals. We conclude that KCC2 may not be an essential component for survival of injured motoneurons and riluzole is able to modulate the intracellular level of calcium and expression of EAAT-2

Motoneuronal inflammasome activation triggers excessive neuroinflammation and impedes regeneration after sciatic nerve injury

Peripheral nerve injuries are accompanied by inflammatory reactions, over-activation of which may hinder recovery. Among pro-inflammatory pathways, inflammasomes are one of the most potent, leading to release of active IL-1β. Our aim was to understand how inflammasomes participate in central inflammatory reactions accompanying peripheral nerve injury.After axotomy of the sciatic nerve, priming and activation of the NLRP3 inflammasome was examined in cells of the spinal cord. Regeneration of the nerve was evaluated after coaptation using sciatic functional index measurements and retrograde tracing.In the first 3 days after the injury, elements of the NLRP3 inflammasome were markedly upregulated in the L4-L5 segments of the spinal cord, followed by assembly of the inflammasome and secretion of active IL-1β. Although glial cells are traditionally viewed as initiators of neuroinflammation, in this acute phase of inflammation, inflammasome activation was found exclusively in affected motoneurons of the ventral horn in our model. This process was significantly inhibited by 5-BDBD, a P2X4 receptor inhibitor and MCC950, a potent NLRP3 inhibitor. Although at later time points the NLRP3 protein was upregulated in microglia too, no signs of inflammasome activation were detected in these cells. Inhibition of inflammasome activation in motoneurons in the first days after nerve injury hindered development of microgliosis in the spinal cord. Moreover, P2X4 or inflammasome inhibition in the acute phase significantly enhanced nerve regeneration on both the morphological and the functional levels.Our results indicate that the central reaction initiated by sciatic nerve injury starts with inflammasome activation in motoneurons of the ventral horn, which triggers a complex inflammatory reaction and activation of microglia. Inhibition of neuronal inflammasome activation not only leads to a significant reduction of microgliosis, but has a beneficial effect on the recovery as well

The effect of grafted neuroectodermal stem cells on injured spinal motoneurons following ventral root avulsion and reimplantation: insights into the molecular mechanism

Avulsion of one or more ventral roots from the spinal cord leads to the death of the majority of affected motoneurons. This process is due to a cascade of events involving activation of astrocytes and microglial cells and the excessive amounts of excitotoxic glutamate release in the injured cord. The aim of the present study was to analyse and compare the therapeutic potential of transplanted NE-GFP-4C murine neuroectodermal cells applied in topically different transplantation paradigms and determine the factors responsible for the motoneuron-rescuing effect. The lumbar 4 (L4) ventral root of Sprague-Dawley rats was avulsed and reimplanted ventrolaterally into the injured cord. Neuroectodermal stem cells were injected immediately following avulsion injury into the L4 segment, into the reimplanted ventral root or were placed in fibrin clot around the reimplanted root. Three months after the primary surgery the L4 motoneuron pool was retrogradely labelled with Fast Blue and the numbers of reinnervating motoneurons were determined. Expression of various factors expected to prevent motoneuron death in the grafted cord was determined by PCR and immunohistochemistry in short term experiments. Animals that received intraspinal stem cell grafts have 70% of their L4 motoneurons regenerated into the vacated endoneural sheaths of the reimplanted root. Morphological reinnervation was accompanied by significant functional recovery. Intraradicular neural stem cell grafting (transplantation into the reimplanted root) resulted in good morphological and functional reinnervation, while both negative controls and animals with perineural stem cell treatment showed poor motor recovery. Stem cell grafts produced the modulatory cytokines IL-1-alpha, IL-6, IL-10, TNF-alpha and MIP-1-alpha, but no neurotrophic factors. The neurons and astrocytes in the ventral horn of grafted animals also produced IL-6 and MIP-1-alpha. The infusion of function-blocking antibodies against all cytokines into the grafted cords completely abolished their motoneuron-rescuing effect, while neutralization of only IL-10 suggested its strong effectivity as concerns motoneuron survival and a milder effect on reinnervation. In this study we have provided evidence that significant numbers of motoneurons can be rescued both by intraradicular and intraspinal stem cell grafting. The pro-inflammatory and anti-inflammatory cytokines selectively secreted by grafted stem cells act in concert to save motoneurons and to promote reinnervation of the target muscles

Neuroectodermal stem cells grafted into the injured spinal cord induce both axonal regeneration and morphological restoration via multiple mechanisms

Spinal cord contusion injury leads to severe loss of grey and white matter and subsequent deficit of motor and sensory functions below the lesion. In this study we investigated whether application of murine clonal embryonic neuroectodermal stem cells can prevent the spinal cord secondary damage and induce functional recovery. Stem cells (NE-GFP-4C cell line) were grafted intraspinally or intravenously immediately or one week after thoracic spinal cord contusion injury. Control animals received cell culture medium or fibrin intraspinally one week after injury. Functional tests (BBB, CatWalk) and detailed morphological analysis were performed to evaluate the effects of grafted cells. Stem cells applied either locally or intravenously induced significantly improved functional recovery compared with their controls. Morphologically, stem cell grafting prevented the formation of secondary injury and promoted sparing of the grey and white matters. The transplanted cells integrated into the host tissue and differentiated into neurons, astrocytes and oligodendrocytes. In intraspinally grafted animals the corticospinal tract axons regenerated along the ventral border of the cavity and have grown several millimeters, even beyond the caudal end of the lesion. The extent of regeneration and functional improvement was inversely related to the amounts of chondroitin sulphate and ephrin-B2 molecules around the cavity and to the microglial and astrocytic reactions in the injured segment early after injury. The grafts produced GDNF, MIP-1a, IL-6 and IL-10 in a paracrine fashion for at least one week. Treating the grafted cords with neutralizing antibodies against these four factors through the use of osmotic pumps nearly completely abolished the effect of the graft. The non-significant functional improvement after function blocking, likely due to the stem cell derivatives settled in the injured cord. These data suggest that grafted neuroectodermal stem cells are able to prevent the secondary spinal cord damage and induce significant regeneration via multiple mechanisms