Restoration of Motor Function through Delayed Intraspinal Delivery of Human IL-10-Encoding Nucleoside-Modified mRNA after Spinal Cord Injury.

Efficient in vivo delivery of anti-inflammatory proteins to modulate the microenvironment of an injured spinal cord and promote neuroprotection and functional recovery is a great challenge. Nucleoside-modified messenger RNA (mRNA) has become a promising new modality that can be utilized for the safe and efficient delivery of therapeutic proteins. Here, we used lipid nanoparticle (LNP)-encapsulated human interleukin-10 (hIL-10)-encoding nucleoside-modified mRNA to induce neuroprotection and functional recovery following rat spinal cord contusion injury. Intralesional administration of hIL-10 mRNA-LNP to rats led to a remarkable reduction of the microglia/macrophage reaction in the injured spinal segment and induced significant functional recovery compared to controls. Furthermore, hIL-10 mRNA treatment induced increased expression in tissue inhibitor of matrix metalloproteinase 1 and ciliary neurotrophic factor levels in the affected spinal segment indicating a time-delayed secondary effect of IL-10 5 d after injection. Our results suggest that treatment with nucleoside-modified mRNAs encoding neuroprotective factors is an effective strategy for spinal cord injury repair.

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

BACKGROUND: 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-1ß. Our aim was to understand how inflammasomes participate in central inflammatory reactions accompanying peripheral nerve injury. METHODS: 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. RESULTS: 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-1ß. 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. CONCLUSIONS: 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 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.

Diazoxide blocks or reduces microgliosis when applied prior or subsequent to motor neuron injury in mice.

Diazoxide (DZX), an anti-hypertonic and anti-hypoglycemic drug, was shown to have anti-inflammatory effects in several injured cell types outside the central nervous system. In the brain, the neuroprotective potential of DZX is well described, however, its anticipated anti-inflammatory effect after acute injury has not been systematically analyzed. To disclose the anti-inflammatory effect of DZX in the central nervous system, an injury was induced in the hypoglossal and facial nuclei and in the oculomotor nucleus by unilateral axonal transection and unilateral target deprivation (enucleation), respectively. On the fourth day after surgery, microglial analysis was performed on tissue in which microglia were DAB-labeled and motoneurons were labeled with immunofluorescence. DZX treatment was given either prophylactically, starting 7 days prior to the injury and continuing until the animals were sacrificed, or postoperatively only, with daily intraperitoneal injections (1.25 mg/kg; in 10 mg/ml dimethyl sulfoxide in distilled water). Prophylactically + postoperatively applied DZX completely eliminated the microglial reaction in each motor nuclei. If DZX was applied only postoperatively, some microglial activation could be detected, but its magnitude was still significantly smaller than the non-DZX-treated controls. The effect of DZX could also be demonstrated through an extended period, as tested in the hypoglossal nucleus on day 7 after the operation. Neuronal counts, determined at day 4 after the operation in the hypoglossal nucleus, demonstrated no loss of motor neurons, however, an increased Feret's diameter of mitochondria could be measured, suggesting increased oxidative stress in the injured cells. The increase of mitochondrial Feret's diameter could also be prevented with DZX treatment.