The pathogenic role of c-Kit+ mast cells in the spinal motor neuron-vascular niche in ALS.

Degeneration of motor neurons, glial cell reactivity, and vascular alterations in the CNS are important neuropathological features of amyotrophic lateral sclerosis (ALS). Immune cells trafficking from the blood also infiltrate the affected CNS parenchyma and contribute to neuroinflammation. Mast cells (MCs) are hematopoietic-derived immune cells whose precursors differentiate upon migration into tissues. Upon activation, MCs undergo degranulation with the ability to increase vascular permeability, orchestrate neuroinflammation and modulate the neuroimmune response. However, the prevalence, pathological significance, and pharmacology of MCs in the CNS of ALS patients remain largely unknown. In autopsy ALS spinal cords, we identified for the first time that MCs express c-Kit together with chymase, tryptase, and Cox-2 and display granular or degranulating morphology, as compared with scarce MCs in control cords. In ALS, MCs were mainly found in the niche between spinal motor neuron somas and nearby microvascular elements, and they displayed remarkable pathological abnormalities. Similarly, MCs accumulated in the motor neuron-vascular niche of ALS murine models, in the vicinity of astrocytes and motor neurons expressing the c-Kit ligand stem cell factor (SCF), suggesting an SCF/c-Kit-dependent mechanism of MC differentiation from precursors. Mechanistically, we provide evidence that fully differentiated MCs in cell cultures can be generated from the murine ALS spinal cord tissue, further supporting the presence of c-Kit+ MC precursors. Moreover, intravenous administration of bone marrow-derived c-Kit+ MC precursors infiltrated the spinal cord in ALS mice but not in controls, consistent with aberrant trafficking through a defective microvasculature. Pharmacological inhibition of c-Kit with masitinib in ALS mice reduced the MC number and the influx of MC precursors from the periphery. Our results suggest a previously unknown pathogenic mechanism triggered by MCs in the ALS motor neuron-vascular niche that might be targeted pharmacologically.

A Nitroalkene Benzoic Acid Derivative Targets Reactive Microglia and Prolongs Survival in an Inherited Model of ALS via NF-κB Inhibition.

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1G93A rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1G93A rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1G93A rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.

Schwann cells orchestrate peripheral nerve inflammation through the expression of CSF1, IL-34, and SCF in amyotrophic lateral sclerosis.

Distal axonopathy is a recognized pathological feature of amyotrophic lateral sclerosis (ALS). In the peripheral nerves of ALS patients, motor axon loss elicits a Wallerian-like degeneration characterized by denervated Schwann cells (SCs) together with immune cell infiltration. However, the pathogenic significance of denervated SCs accumulating following impaired axonal growth in ALS remains unclear. Here, we analyze SC phenotypes in sciatic nerves of ALS patients and paralytic SOD1G93A rats, and identify remarkably similar and specific reactive SC phenotypes based on the pattern of S100ß, GFAP, isolectin and/or p75NTR immunoreactivity. Different subsets of reactive SCs expressed colony-stimulating factor-1 (CSF1) and Interleukin-34 (IL-34) and closely interacted with numerous endoneurial CSF-1R-expressing monocyte/macrophages, suggesting a paracrine mechanism of myeloid cell expansion and activation. SCs bearing phagocytic phenotypes as well as endoneurial macrophages expressed stem cell factor (SCF), a trophic factor that attracts and activates mast cells through the c-Kit receptor. Notably, a subpopulation of Ki67+ SCs expressed c-Kit in the sciatic nerves of SOD1G93A rats, suggesting a signaling pathway that fuels SC proliferation in ALS. c-Kit+ mast cells were also abundant in the sciatic nerve from ALS donors but not in controls. Pharmacological inhibition of CSF-1R and c-Kit with masitinib in SOD1G93A rats potently reduced SC reactivity and immune cell infiltration in the sciatic nerve and ventral roots, suggesting a mechanism by which the drug ameliorates peripheral nerve pathology. These findings provide strong evidence for a previously unknown inflammatory mechanism triggered by SCs in ALS peripheral nerves that has broad application in developing novel therapies.

CD34 Identifies a Subset of Proliferating Microglial Cells Associated with Degenerating Motor Neurons in ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons accompanied by proliferation of reactive microglia in affected regions. However, it is unknown whether the hematopoietic marker CD34 can identify a subpopulation of proliferating microglial cells in the ALS degenerating spinal cord. Immunohistochemistry for CD34 and microglia markers was performed in lumbar spinal cords of ALS rats bearing the SOD1G93A mutation and autopsied ALS and control human subjects. Characterization of CD34-positive cells was also performed in primary cell cultures of the rat spinal cords. CD34 was expressed in a large number of cells that closely interacted with degenerating lumbar spinal cord motor neurons in symptomatic SOD1G93A rats, but not in controls. Most CD34+ cells co-expressed the myeloid marker CD11b, while only a subpopulation was stained for Iba1 or CD68. Notably, CD34+ cells actively proliferated and formed clusters adjacent to damaged motor neurons bearing misfolded SOD1. CD34+ cells were identified in the proximity of motor neurons in autopsied spinal cord from sporadic ALS subjects but not in controls. Cell culture of symptomatic SOD1G93A rat spinal cords yielded a large number of CD34+ cells exclusively in the non-adherent phase, which generated microglia after successive passaging. A yet unrecognized CD34+ cells, expressing or not the microglial marker Iba1, proliferate and accumulate adjacent to degenerating spinal motor neurons, representing an intriguing cell target for approaching ALS pathogenesis and therapeutics.

Mast cells and neutrophils mediate peripheral motor pathway degeneration in ALS.

Neuroinflammation is a recognized pathogenic mechanism underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS), but the inflammatory mechanisms influencing peripheral motor axon degeneration remain largely unknown. A recent report showed a pathogenic role for c-Kit-expressing mast cells mediating inflammation and neuromuscular junction denervation in muscles from SOD1G93A rats. Here, we have explored whether mast cells infiltrate skeletal muscles in autopsied muscles from ALS patients. We report that degranulating mast cells were abundant in the quadriceps muscles from ALS subjects but not in controls. Mast cells were associated with myofibers and motor endplates and, remarkably, interacted with neutrophils forming large extracellular traps. Mast cells and neutrophils were also abundant around motor axons in the extensor digitorum longus muscle, sciatic nerve, and ventral roots of symptomatic SOD1G93A rats, indicating that immune cell infiltration extends along the entire peripheral motor pathway. Postparalysis treatment of SOD1G93A rats with the tyrosine kinase inhibitor drug masitinib prevented mast cell and neutrophil infiltration, axonal pathology, secondary demyelination, and the loss of type 2B myofibers, compared with vehicle-treated rats. These findings provide further evidence for a yet unrecognized contribution of immune cells in peripheral motor pathway degeneration that can be therapeutically targeted by tyrosine kinase inhibitors.