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

Passive transfer of blood sera from ALS patients with identified mutations results in elevated motoneuronal calcium level and loss of motor neurons in the spinal cord of mice

Introduction: Previously, we demonstrated the degeneration of axon terminals in mice after repeated injections of blood sera from amyotrophic lateral sclerosis (ALS) patients with identified mutations. However, whether a similar treatment affects the cell body of motor neurons (MNs) remained unresolved. Methods: Sera from healthy individuals or ALS patients with a mutation in different ALS-related genes were intraperitoneally injected into ten-week-old male Balb/c mice (n = 3/serum) for two days. Afterward, the perikaryal calcium level was measured using electron microscopy. Furthermore, the optical disector method was used to evaluate the number of lumbar MNs. Results: The cytoplasmic calcium level of the lumbar MNs of the ALS-serum-treated mice, compared to untreated and healthy-serum-treated controls, was significantly elevated. While injections of the healthy serum did not reduce the number of MNs compared to the untreated control group, ALS sera induced a remarkable loss of MNs. Discussion: Similarly to the distant motor axon terminals, the injection of blood sera of ALS patients has a rapid degenerative effect on MNs. Analogously, the magnitude of the evoked changes was specific to the type of mutation; furthermore, the degeneration was most pronounced in the group treated with sera from ALS patients with a mutation in the chromosome 9 open reading frame 72 gene

Passive transfer of sera from als patients with identified mutations evokes an increased synaptic vesicle number and elevation of calcium levels in motor axon terminals, similar to sera from sporadic patients

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role

Upregulation of Nucleotide-Binding Oligomerization Domain-, LRR- and Pyrin Domain-Containing Protein 3 in Motoneurons Following Peripheral Nerve Injury in Mice

Neuronal injuries are accompanied by release and accumulation of damage-associated molecules, which in turn may contribute to activation of the immune system. Since a wide range of danger signals (including endogenous ones) are detected by the nucleotide-binding oligomerization domain-, LRR- and pyrin domain-containing protein 3 (NLRP3) pattern recognition receptor, we hypothesized that NLRP3 may become activated in response to motor neuron injury. Here we show that peripheral injury of the oculomotor and the hypoglossal nerves results in upregulation of NLRP3 in corresponding motor nuclei in the brainstem of mice. Although basal expression of NLRP3 was observed in microglia, astroglia and neurons as well, its upregulation and co-localization with apoptosis-associated speck-like protein containing a caspase activation and recruitment domain, suggesting inflammasome activation, was only detected in neurons. Consequently, increased production of active pro-inflammatory cytokines interleukin-1 beta and interleukin-18 were detected after hypoglossal nerve axotomy. Injury-sensitive hypoglossal neurons responded with a more pronounced NLRP3 upregulation than injury-resistant motor neurons of the oculomotor nucleus. We further demonstrated that the mitochondrial protector diazoxide was able to reduce NLRP3 upregulation in a post-operative treatment paradigm. Our results indicate that NLRP3 is activated in motoneurons following acute nerve injury. Blockade of NLRP3 activation might contribute to the previously observed anti-inflammatory and neuroprotective effects of diazoxide

Inflammasome activation in peritumoral astrocytes is a key player in breast cancer brain metastasis development

Inflammasomes, primarily responsible for the activation of IL-1β, have emerged as critical regulators of the tumor microenvironment. By using in vivo and in vitro brain metastasis models, as well as human samples to study the role of the NLRP3 inflammasome in triple-negative breast cancer (TNBC) brain metastases, we found NLRP3 inflammasome components and IL-1β to be highly and specifically expressed in peritumoral astrocytes. Soluble factors from TNBC cells induced upregulation and activation of NLRP3 and IL-1β in astrocytes, while astrocyte-derived mediators augmented the proliferation of metastatic cells. In addition, inhibition of NLRP3 inflammasome activity using MCC950 or dampening the downstream effect of IL-1β prevented the proliferation increase in cancer cells. In vivo, MCC950 reduced IL-1β expression in peritumoral astrocytes, as well as the levels of inflammasome components and active IL-1β. Most importantly, significantly retarded growth of brain metastatic tumors was observed in mice treated with MCC950. Overall, astrocytes contribute to TNBC progression in the brain through activation of the NLRP3 inflammasome and consequent IL-1β release. We conclude that pharmacological targeting of inflammasomes may become a novel strategy in controlling brain metastatic diseases