The Astrocytic S100B Protein with Its Receptor RAGE Is Aberrantly Expressed in SOD1(G93A) Models, and Its Inhibition Decreases the Expression of Proinflammatory Genes

Neuroinflammation is one of the major players in amyotrophic lateral sclerosis (ALS) pathogenesis, and astrocytes are significantly involved in this process. The astrocytic protein S100B can be released in pathological states activating the receptor for advanced glycation end products (RAGE). Different indications point to an aberrant expression of S100B and RAGE in ALS. In this work, we observed that S100B and RAGE are progressively and selectively upregulated in astrocytes of diseased rats with a tissue-specific timing pattern, correlated to the level of neurodegeneration. The expression of the full-length and soluble RAGE isoforms could also be linked to the degree of tissue damage. The mere presence of mutant SOD1 is able to increase the intracellular levels and release S100B from astrocytes, suggesting the possibility that an increased astrocytic S100B expression might be an early occurring event in the disease. Finally, our findings indicate that the protein may exert a proinflammatory role in ALS, since its inhibition in astrocytes derived from SOD1(G93A) mice limits the expression of reactivity-linked/proinflammatory genes. Thus, our results propose the S100B-RAGE axis as an effective contributor to the pathogenesis of the disease, suggesting its blockade as a rational target for a therapeutic intervention in ALS

N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X3 receptor (Asn139, Asn170, Asn194 and Asn290) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn170 is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr172 in the same glycosylation consensus. Asn194 and Asn290 are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn170 mutation or the Asn139/Asn290 double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X3 mutants where residue Asn170 is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X3 recepto

The S100A4 Transcriptional Inhibitor Niclosamide Reduces Pro-Inflammatory and Migratory Phenotypes of Microglia: Implications for Amyotrophic Lateral Sclerosis

S100A4, belonging to a large multifunctional S100 protein family, is a Ca2+-binding protein with a significant role in stimulating the motility of cancer and immune cells, as well as in promoting pro-inflammatory properties in different cell types. In the CNS, there is limited information concerning S100A4 presence and function. In this study, we analyzed the expression of S100A4 and the effect of the S100A4 transcriptional inhibitor niclosamide in murine activated primary microglia. We found that S100A4 was strongly up-regulated in reactive microglia and that niclosamide prevented NADPH oxidase 2, mTOR (mammalian target of rapamycin), and NF-kappa B (nuclear factor-kappa B) increase, cytoskeletal rearrangements, migration, and phagocytosis. Furthermore, we found that S100A4 was significantly up-regulated in astrocytes and microglia in the spinal cord of a transgenic rat SOD1-G93A model of amyotrophic lateral sclerosis. Finally, we demonstrated the increased expression of S100A4 also in fibroblasts derived from amyotrophic lateral sclerosis (ALS) patients carrying SOD1 pathogenic variants. These results ascribe S100A4 as a marker of microglial reactivity, suggesting the contribution of S100A4-regulated pathways to neuroinflammation, and identify niclosamide as a possible drug in the control and attenuation of reactive phenotypes of microglia, thus opening the way to further investigation for a new application in neurodegenerative conditions

Frataxin deficiency shifts metabolism to promote reactive microglia via glucose catabolism

Immunometabolism investigates the intricate relationship between the immune system and cellular metabolism. This study delves into the consequences of mitochondrial frataxin (FXN) depletion, the primary cause of Friedreich's ataxia (FRDA), a debilitating neurodegenerative condition characterized by impaired coordination and muscle control. By using single-cell RNA sequencing, we have identified distinct cellular clusters within the cerebellum of an FRDA mouse model, emphasizing a significant loss in the homeostatic response of microglial cells lacking FXN. Remarkably, these microglia deficient in FXN display heightened reactive responses to inflammatory stimuli. Furthermore, our metabolomic analyses reveal a shift towards glycolysis and itaconate production in these cells. Remarkably, treatment with butyrate counteracts these immunometabolic changes, triggering an antioxidant response via the itaconate-Nrf2-GSH pathways and suppressing the expression of inflammatory genes. Furthermore, we identify Hcar2 (GPR109A) as a mediator involved in restoring the homeostasis of microglia without FXN. Motor function tests conducted on FRDA mice underscore the neuroprotective attributes of butyrate supplementation, enhancing neuromotor performance. In conclusion, our findings elucidate the role of disrupted homeostatic function in cerebellar microglia in the pathogenesis of FRDA. Moreover, they underscore the potential of butyrate to mitigate inflammatory gene expression, correct metabolic imbalances, and improve neuromotor capabilities in FRDA

Inflammation in the CNS and PNS: From Molecular Basis to Therapy

Our understanding of the pathophysiology of the nervous system has advanced significantly in the last few years, but there are still many unanswered questions [...]

Neuroinflammation in Friedreich's Ataxia

Friedreich's ataxia (FRDA) is a rare genetic disorder caused by mutations in the gene frataxin, encoding for a mitochondrial protein involved in iron handling and in the biogenesis of iron-sulphur clusters, and leading to progressive nervous system damage. Although the overt manifestations of FRDA in the nervous system are mainly observed in the neurons, alterations in non-neuronal cells may also contribute to the pathogenesis of the disease, as recently suggested for other neurodegenerative disorders. In FRDA, the involvement of glial cells can be ascribed to direct effects caused by frataxin loss, eliciting different aberrant mechanisms. Iron accumulation, mitochondria dysfunction, and reactive species overproduction, mechanisms identified as etiopathogenic in neurons in FRDA, can similarly affect glial cells, leading them to assume phenotypes that can concur to and exacerbate neuron loss. Recent findings obtained in FRDA patients and cellular and animal models of the disease have suggested that neuroinflammation can accompany and contribute to the neuropathology. In this review article, we discuss evidence about the involvement of neuroinflammatory-related mechanisms in models of FRDA and provide clues for the modulation of glial-related mechanisms as a possible strategy to improve disease features

Pathways of survival induced by NGF and extracellular ATP after growth factor deprivation

In a previous work we demonstrated that extracellular adenosine-5'-triphosphate (ATP), acting on P2 receptors, exerts neuritogenic and trophic effects on the phaeochromocytoma PC12 cell line. These actions are comparable to those sustained by nerve growth factor (NGF) and involve several overlapping pathways. In this work, we describe some of the mechanisms recruited by ATP and NGF in maintaining PC12 cell survival after serum deprivation. We show that both ATP and NGF upregulate the expression of the stress-induced heat shock protein HSP70 and HSP90, whilst glucose-response protein GRP75 and GRP78 are not affected. In parallel with NGF, ATP prevents the cleavage and activation of caspase-2 and inhibits the release of cytochrome c from mitochondria into the cytoplasm. Finally, neither NGF, nor ATP directly modulate the expression of P2 receptors in the induction of cell survival. Our data contribute to dissect the biological mechanisms activated by extracellular purines exerting trophic actions and to establish that survival and neurite outgrowth lie on different mechanistic pathways

The Astrocytic S100B Protein with Its Receptor RAGE Is Aberrantly Expressed in SOD1G93AModels, and Its Inhibition Decreases the Expression of Proinflammatory Genes

Neuroinflammation is one of the major players in amyotrophic lateral sclerosis (ALS) pathogenesis, and astrocytes are significantly involved in this process. The astrocytic protein S100B can be released in pathological states activating the receptor for advanced glycation end products (RAGE). Different indications point to an aberrant expression of S100B and RAGE in ALS. In this work, we observed that S100B and RAGE are progressively and selectively upregulated in astrocytes of diseased rats with a tissue-specific timing pattern, correlated to the level of neurodegeneration. The expression of the full-length and soluble RAGE isoforms could also be linked to the degree of tissue damage. The mere presence of mutant SOD1 is able to increase the intracellular levels and release S100B from astrocytes, suggesting the possibility that an increased astrocytic S100B expression might be an early occurring event in the disease. Finally, our findings indicate that the protein may exert a proinflammatory role in ALS, since its inhibition in astrocytes derived from SOD1G93Amice limits the expression of reactivity-linked/proinflammatory genes. Thus, our results propose the S100B-RAGE axis as an effective contributor to the pathogenesis of the disease, suggesting its blockade as a rational target for a therapeutic intervention in ALS

Microglial Pruning: Relevance for Synaptic Dysfunction in Multiple Sclerosis and Related Experimental Models

Microglia, besides being able to react rapidly to a wide range of environmental changes, are also involved in shaping neuronal wiring. Indeed, they actively participate in the modulation of neuronal function by regulating the elimination (or "pruning") of weaker synapses in both physiologic and pathologic processes. Mounting evidence supports their crucial role in early synaptic loss, which is emerging as a hallmark of several neurodegenerative diseases, including multiple sclerosis (MS) and its preclinical models. MS is an inflammatory, immune-mediated pathology of the white matter in which demyelinating lesions may cause secondary neuronal death. Nevertheless, primitive grey matter (GM) damage is emerging as an important contributor to patients' long-term disability, since it has been associated with early and progressive cognitive decline (CD), which seriously worsens the quality of life of MS patients. Widespread synapse loss even in the absence of demyelination, axon degeneration and neuronal death has been demonstrated in different GM structures, thus raising the possibility that synaptic dysfunction could be an early and possibly independent event in the neurodegenerative process associated with MS. This review provides an overview of microglial-dependent synapse elimination in the neuroinflammatory process that underlies MS and its experimental models