Ferroptosis Mediates Cuprizone-Induced Loss of Oligodendrocytes and Demyelination.

Multiple sclerosis (MS) is a chronic demyelinating disease of the CNS. Cuprizone (CZ), a copper chelator, is widely used to study demyelination and remyelination in the CNS, in the context of MS. However, the mechanisms underlying oligodendrocyte (OL) cell loss and demyelination are not known. As copper-containing enzymes play important roles in iron homeostasis and controlling oxidative stress, we examined whether chelating copper leads to disruption of molecules involved in iron homeostasis that can trigger iron-mediated OL loss. We show that giving mice (male) CZ in the diet induces rapid loss of OL in the corpus callosum by 2 d, accompanied by expression of several markers for ferroptosis, a relatively newly described form of iron-mediated cell death. In ferroptosis, iron-mediated free radicals trigger lipid peroxidation under conditions of glutathione insufficiency, and a reduced capacity to repair lipid damage. This was further confirmed using a small-molecule inhibitor of ferroptosis that prevents CZ-induced loss of OL and demyelination, providing clear evidence of a copper-iron connection in CZ-induced neurotoxicity. This work has wider implications for disorders, such as multiple sclerosis and CNS injury.SIGNIFICANCE STATEMENT Cuprizone (CZ) is a copper chelator that induces demyelination. Although it is a widely used model to study demyelination and remyelination in the context of multiple sclerosis, the mechanisms mediating demyelination is not fully understood. This study shows, for the first time, that CZ induces demyelination via ferroptosis-mediated rapid loss of oligodendrocytes. This work shows that chelating copper with CZ leads to the expression of molecules that rapidly mobilize iron from ferritin (an iron storage protein), that triggers iron-mediated lipid peroxidation and oligodendrocyte loss (via ferroptosis). Such rapid mobilization of iron from cellular stores may also play a role in cell death in other neurologic conditions.

Lysophosphatidic acid receptor type 2 activation contributes to secondary damage after spinal cord injury in mice.

Lysophosphatidic acid (LPA) is an extracellular lipid mediator involved in many physiological functions by signaling through six known G-protein-coupled receptors (LPA1-LPA6). In the central nervous system (CNS), LPA mediates a wide range of effects, including neural progenitor cell physiology, astrocyte and microglia activation, neuronal cell death, axonal retraction, and contributions to pain, schizophrenia and hydrocephalus. We recently reported that LPA-LPA1 signaling mediates functional deficits and myelin loss after spinal cord injury (SCI). Here, we provide clear evidence on the deleterious contribution of another LPA receptor, LPA2, to myelin loss after SCI. We found that LPA2 is constitutively expressed in the spinal cord parenchyma and its transcripts were up-regulated after contusion injury, in part, by microglial cells. We also found that the demyelinating lesion triggered by intraspinal injection of LPA into the undamaged spinal cord was markedly reduced in the lack of LPA2. Similarly, LPA2 deficient mice showed enhanced motor skills and myelin sparing after SCI. To gain insights into the detrimental actions of LPA2 in spinal cord we performed cell culture studies. These experiments revealed that, similar to LPA1, activation of microglia LPA2 led to oligodendrocyte cell death. Moreover, we also found that the cytotoxic effects underlaying microglial LPA-LPA2 axis were mediated by the release of purines by microglia and the activation of P2X7 receptor on oligodendrocytes. Overall, this study provides new mechanistic insights into how LPA contributes to SCI physiopathology, and suggest that targeting LPA2 could be a novel therapeutic approach for the treatment of acute SCI.

Beneficial effects of IL-37 after spinal cord injury in mice.

IL-37, a member of the IL-1 family, broadly reduces innate inflammation as well as acquired immunity. Whether the antiinflammatory properties of IL-37 extend to the central nervous system remains unknown, however. In the present study, we subjected mice transgenic for human IL-37 (hIL-37tg) and wild-type (WT) mice to spinal cord contusion injury and then treated them with recombinant human IL-37 (rIL-37). In the hIL-37tg mice, the expression of IL-37 was barely detectable in the uninjured cords, but was strongly induced at 24 h and 72 h after the spinal cord injury (SCI). Compared with WT mice, hIL-37tg mice exhibited increased myelin and neuronal sparing and protection against locomotor deficits, including 2.5-fold greater speed in a forced treadmill challenge. Reduced levels of cytokines (e.g., an 80% reduction in IL-6) were observed in the injured cords of hIL-37tg mice, along with lower numbers of blood-borne neutrophils, macrophages, and activated microglia. We treated WT mice with a single intraspinal injection of either full-length or processed rIL-37 after the injury and found that the IL-37-treated mice had significantly enhanced locomotor skills in an open field using the Basso Mouse Scale, as well as supported faster speed on a mechanical treadmill. Treatment with both forms of rIL-37 led to similar beneficial effects on locomotor recovery after SCI. This study presents novel data indicating that IL-37 suppresses inflammation in a clinically relevant model of SCI, and suggests that rIL-37 may have therapeutic potential for the treatment of acute SCI.

Maresin 1 Promotes Inflammatory Resolution, Neuroprotection, and Functional Neurological Recovery After Spinal Cord Injury.

Resolution of inflammation is defective after spinal cord injury (SCI), which impairs tissue integrity and remodeling and leads to functional deficits. Effective pharmacological treatments for SCI are not currently available. Maresin 1 (MaR1) is a highly conserved specialized proresolving mediator (SPM) hosting potent anti-inflammatory and proresolving properties with potent tissue regenerative actions. Here, we provide evidence that the inappropriate biosynthesis of SPM in the lesioned spinal cord hampers the resolution of inflammation and leads to deleterious consequences on neurological outcome in adult female mice. We report that, after spinal cord contusion injury in adult female mice, the biosynthesis of SPM is not induced in the lesion site up to 2 weeks after injury. Exogenous administration of MaR1, a highly conserved SPM, propagated inflammatory resolution after SCI, as revealed by accelerated clearance of neutrophils and a reduction in macrophage accumulation at the lesion site. In the search of mechanisms underlying the proresolving actions of MaR1 in SCI, we found that this SPM facilitated several hallmarks of resolution of inflammation, including reduction of proinflammatory cytokines (CXCL1, CXCL2, CCL3, CCL4, IL6, and CSF3), silencing of major inflammatory intracellular signaling cascades (STAT1, STAT3, STAT5, p38, and ERK1/2), redirection of macrophage activation toward a prorepair phenotype, and increase of the phagocytic engulfment of neutrophils by macrophages. Interestingly, MaR1 administration improved locomotor recovery significantly and mitigated secondary injury progression in a clinical relevant model of SCI. These findings suggest that proresolution, immunoresolvent therapies constitute a novel approach to improving neurological recovery after acute SCI.SIGNIFICANCE STATEMENT Inflammation is a protective response to injury or infection. To result in tissue homeostasis, inflammation has to resolve over time. Incomplete or delayed resolution leads to detrimental effects, including propagated tissue damage and impaired wound healing, as occurs after spinal cord injury (SCI). We report that inflammation after SCI is dysregulated in part due to inappropriate synthesis of proresolving lipid mediators. We demonstrate that the administration of the resolution agonist referred to as maresin 1 (MaR1) after SCI actively propagates resolution processes at the lesion site and improves neurological outcome. MaR1 is identified as an interventional candidate to attenuate dysregulated lesional inflammation and to restore functional recovery after SCI.

Role of IL-10 in Resolution of Inflammation and Functional Recovery after Peripheral Nerve Injury.

A rapid proinflammatory response after peripheral nerve injury is required for clearance of tissue debris (Wallerian degeneration) and effective regeneration. Unlike the CNS, this response is rapidly terminated in peripheral nerves starting between 2 and 3 weeks after crush injury. We examined the expression and role of the anti-inflammatory cytokine IL-10 in the resolution of inflammation and regeneration after sciatic nerve crush injury in mice. IL-10 mRNA increased over the first 7 d after injury, whereas at the protein level, immunofluorescence labeling showed IL-10(+) cells increased almost 3-fold in the first 3 weeks, with macrophages being the major cell type expressing IL-10. The role of IL-10 in nerve injury was assessed using IL-10-null mice. Increased numbers of macrophages were found in the distal segment of IL-10-null mice at early (3 d) and late (14 and 21 d) time points, suggesting that IL-10 may play a role in controlling the early influx and the later efflux of macrophages out of the nerve. A chemokine/cytokine PCR array of the nerve 24 h after crush showed a 2- to 4-fold increase in the expression of 10 proinflammatory mediators in IL-10(-/-) mice. In addition, myelin phagocytosis in vitro by LPS stimulated bone-marrow-derived macrophages from IL-10-null mice failed to downregulate expression of proinflammatory chemokines/cytokines, suggesting that IL-10 is required for the myelin-phagocytosis-induced shift of macrophages from proinflammatory to anti-inflammatory/pro-repair phenotype. The failure to switch off inflammation in IL-10-null mice was accompanied by impaired axon regeneration and poor recovery of motor and sensory function. SIGNIFICANCE STATEMENT: An appropriately regulated inflammatory response after peripheral nerve injury is essential for axon regeneration and recovery. The aim of this study was to investigate the expression and role of the anti-inflammatory cytokine IL-10 in terminating inflammation after sciatic nerve crush injury and promoting regeneration. IL-10 is rapidly expressed by macrophages after crush injury. Its role was assessed using IL-10-null mice, which showed that IL-10 plays a role in controlling the early influx and the later efflux of macrophages out of the injured nerve, reduces the expression of proinflammatory chemokines and cytokines, and is required for myelin-phagocytosis-induced shift of macrophages from proinflammatory to anti-inflammatory. Furthermore, lack of IL-10 leads to impaired axon regeneration and poor recovery of motor and sensory function.

Activation of Lysophosphatidic Acid Receptor Type 1 Contributes to Pathophysiology of Spinal Cord Injury.

Lysophosphatidic acid (LPA) is an extracellular lipid mediator involved in many physiological functions that signals through six known G-protein-coupled receptors (LPA1-LPA6). A wide range of LPA effects have been identified in the CNS, including neural progenitor cell physiology, astrocyte and microglia activation, neuronal cell death, axonal retraction, and development of neuropathic pain. However, little is known about the involvement of LPA in CNS pathologies. Herein, we demonstrate for the first time that LPA signaling via LPA1 contributes to secondary damage after spinal cord injury. LPA levels increase in the contused spinal cord parenchyma during the first 14 d. To model this potential contribution of LPA in the spinal cord, we injected LPA into the normal spinal cord, revealing that LPA induces microglia/macrophage activation and demyelination. Use of a selective LPA1 antagonist or mice lacking LPA1 linked receptor-mediated signaling to demyelination, which was in part mediated by microglia. Finally, we demonstrate that selective blockade of LPA1 after spinal cord injury results in reduced demyelination and improvement in locomotor recovery. Overall, these results support LPA-LPA1 signaling as a novel pathway that contributes to secondary damage after spinal cord contusion in mice and suggest that LPA1 antagonism might be useful for the treatment of acute spinal cord injury. SIGNIFICANCE STATEMENT: This study reveals that LPA signaling via LPA receptor type 1 activation causes demyelination and functional deficits after spinal cord injury.

Assessment of focused ultrasound stimulation to induce peripheral nerve activity and potential damage in vivo.

Introduction: Peripheral neuroprostheses are aimed to restore loss of sensory and motor functions by interfacing axons in the peripheral nerves. Most common interfaces in neuroprostheses are electrodes that establish electrical connection with peripheral axons. However, some challenges arise related to long-term functionality, durability, and body response. Recently, focused ultrasound stimulation (FUS) has emerged as a non-invasive approach to modulate the nervous system. However, it is controversial whether FUS can induce axon depolarization. Methods: We have assessed FUS applied in vivo to the rat peripheral nerve, with two objectives: first, to test whether FUS activates peripheral nerves under different stimulation conditions, and second, to evaluate if FUS inflicts damage to the nerve. FUS was delivered with three ultrasound transducers (Sonic Concept H115, H107, and H102) covering the largest set of parameters examined for FUS of peripheral nerves so far. Results: We did not obtain reliable evoked action potentials in either nerves or muscles, under any FUS condition applied, neither over the skin nor directly to the nerve exposed. Additional experiments ex vivo and in vivo on mice, confirmed this conclusion. When FUS stimulation was applied directly to the exposed sciatic nerve, neuromuscular function decreased significantly, and recovered one week later, except for FUS at 0.25 MHz. Histologically, degenerating nerve fibers were observed, with a tendency to be higher with the lower FUS frequency. Discussion: Past reports on the ability of ultrasound to stimulate the peripheral nerve are controversial. After testing a wide range of FUS conditions, we conclude that it is not a reliable and safe method for stimulating the peripheral nerve. Special consideration should be taken, especially when low-frequency FUS is applied, as it may lead to nerve damage.

Beneficial effects of αB-crystallin in spinal cord contusion injury.

αB-crystallin is a member of the heat shock protein family that exerts cell protection under several stress-related conditions. Recent studies have revealed that αB-crystallin plays a beneficial role in a mouse model of multiple sclerosis, brain ischemia, and Alexander disease. Whether αB-crystallin plays a role in modulating the secondary damage after CNS trauma is not known. We report here that αB-crystallin mediates protective effects after spinal cord injury. The levels of αB-crystallin are reduced in spinal cord tissue following contusion lesion. In addition, administration of recombinant human αB-crystallin for the first week after contusion injury leads to sustained improvement in locomotor skills and amelioration of secondary tissue damage. We also provide evidence that recombinant human αB-crystallin modulates the inflammatory response in the injured spinal cord, leading to increased infiltration of granulocytes and reduced recruitment of inflammatory macrophages. Furthermore, the delivery of recombinant human αB-crystallin promotes greater locomotor recovery even when the treatment is initiated 6 h after spinal cord injury. Our findings suggest that administration of recombinant human αB-crystallin may be a good therapeutic approach for treating acute spinal cord injury, for which there is currently no effective treatment.

FGF-2 low molecular weight selectively promotes neuritogenesis of motor neurons in vitro.

In this study, we screened in vitro the different capabilities of trophic factors with promising effect for enhancing selective regeneration and thus promoting specific reinnervation of target organs after peripheral nerve regeneration. We found that FGF-2 (18 kDa) was the trophic factor that exerted the most selective effect in promoting neurite outgrowth of spinal motoneurons both in terms of elongation and arborization. The mechanism underlying this effect on neuritogenesis seems related to FGF-2 enhancing the interaction between FGFR-1 and PSA-NCAM. The interaction of these two receptors is important during the early stages of neuritogenesis and pathfinding, while integrin alpha7B subunit seems to play a role during neurite stabilization.

Bone marrow transplantation in dysferlin-deficient mice results in a mild functional improvement.

Dysferlinopathies are caused by mutations in the DYSF gene. Dysferlin is a protein mainly expressed in the skeletal muscle and monocytes. Cell therapy constitutes a promising tool for the treatment of muscular dystrophies. The aim of our study was to evaluate the effect of bone marrow transplantation (BMT) using the A/J Dysf(prmd) mouse model of dysferlinopathy. For that purpose, we studied dysferlin expression by western blot and/or immunohistochemistry in transplanted mice and controls. Computerized analyses of locomotion and electrophysiological techniques were also performed to test the functional improvement. We observed dysferlin expression in splenocytes, but not in the skeletal muscle of the transplanted mice. However, the locomotion test, electromyography studies, and muscle histology showed an improvement in all transplanted mice that was more significant in the animals transplanted with dysferlin⁺/⁺ cells. In conclusion, although BMT restores dysferlin expression in monocytes, but not in skeletal muscle, muscle function was partially recovered. We propose that the slight improvement observed in the functional studies could be related with factors, such as the hepatocyte growth factor, released after BMT that prevented muscle degeneration.

Randall-Selitto test: a new approach for the detection of neuropathic pain after spinal cord injury.

In this work we assess the usefulness of the Randall-Selitto test as a method to detect and quantify neuropathic pain responses in rats subjected to different spinal cord injuries. The mechanical nociceptive thresholds were significantly reduced during follow-up after spinal cord contusion or transection. Our results demonstrate that the Randall-Selitto test allows the detection of neuropathic pain both in forepaws and hindpaws, as well as in dorsal and plantar surfaces. Moreover, it does not require weight support capacity, so it can be used at early time points after the injury. This is the first time that this method has been used to describe the changes in nociceptive thresholds that take place after spinal cord injuries of different severities over time.

Electrophysiological analysis of a murine model of motoneuron disease.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motoneurons of the primary motor cortex, the brainstem and the spinal cord, for which there are not effective treatments. Several transgenic mice that mimic motoneuron disease have been used to investigate potential treatments. The objective of this work is to characterize electrophysiologically the SOD1(G93A) transgenic mouse model of ALS, and to provide useful markers to improve early detection and monitoring of progression of the disease. METHODS: We performed nerve conduction tests, motor unit number estimation (MUNE), H reflex tests and motor evoked potentials (MEPs) in a cohort of transgenic and wild type mice from 4 to 16 weeks of age. RESULTS: The results revealed dysfunction of spinal motoneurons evidenced by deficits in motor nerve conduction tests starting at 8 weeks of age, earlier in proximal than in distal muscles of the hindlimb. MUNE demonstrated that spinal motoneurons loss muscle innervation and have a deficit in their sprouting capacity. Motor evoked potentials revealed that, coexisting with peripheral deficits, there was a dysfunction of central motor tracts that started also at 8 weeks, indicating progressive dysfunction of upper motoneurons. CONCLUSIONS: These electrophysiological results provide important information about the SOD1(G93A) mouse model, as they demonstrate by the first time alterations of central motor pathways simultaneously to lower motoneuron dysfunction, well before functional abnormalities appear (by 12 weeks of age). SIGNIFICANCE: The finding of concomitant dysfunction of upper and lower motoneurons contributes to the validation of the SOD1(G93A) mouse as model of ALS, because this parallel involvement is a diagnostic condition for ALS. Electrophysiological tests can be used as early markers of the disease and to evaluate the potential benefits of new treatments on both upper and lower motoneurons.