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.

Neuroprotective effects of niclosamide on disease progression via inflammatory pathways modulation in SOD1-G93A and FUS-associated amyotrophic lateral sclerosis models.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease influenced by genetic, epigenetic, and environmental factors, resulting in dysfunction in cellular and molecular pathways. The limited efficacy of current treatments highlights the need for combination therapies targeting multiple aspects of the disease. Niclosamide, an anthelminthic drug listed as an essential medicine, has been repurposed in clinical trials for different diseases due to its anti-inflammatory and anti-fibrotic properties. Niclosamide can inhibit various molecular pathways (e.g., STAT3, mTOR) that are dysregulated in ALS, suggesting its potential to disrupt these altered mechanisms associated with the pathology. We administered niclosamide intraperitoneally to two transgenic murine models, SOD1-G93A and FUS mice, mimicking key pathological processes of ALS. The treatment was initiated at the onset of symptoms, and we assessed disease progression by neurological scores, rotarod and wire tests, and monitored survival. Furthermore, we investigated cellular and molecular mechanisms affected by niclosamide in the spinal cord and muscle of ALS mice. In both models, the administration of niclosamide resulted in a slowdown of disease progression, an increase in survival rates, and an improvement in tissue pathology. This was characterised by reduced gliosis, motor neuron loss, muscle atrophy, and inflammatory pathways. Based on these results, our findings demonstrate that niclosamide can impact multiple pathways involved in ALS. This multi-targeted approach leads to a slowdown in the progression of the disease, positioning niclosamide as a promising candidate for repurposing in the treatment of ALS.

C9orf72 Toxic Species Affect ArfGAP-1 Function.

Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs. We previously reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in the cell nuclei. To further characterize the process, we set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS RNA repeats. Interestingly, pathway analysis showed that the mRNAs involved in membrane trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicated that C9orf72 toxic species affect the membrane trafficking route regulated by ADP-Ribosylation Factor 1 GTPase Activating Protein (ArfGAP-1), which exerts its GTPase-activating function on the small GTPase ADP-ribosylation factor 1 to dissociate coat proteins from Golgi-derived vesicles. We demonstrate that the function of ArfGAP-1 is specifically affected by expanded C9orf72 RNA repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), indicating the retrograde Golgi-to-ER vesicle-mediated transport as a target of C9orf72 toxicity.

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.

The Contribution of Non-Neuronal Cells in Neurodegeneration: From Molecular Pathogenesis to Therapeutic Challenges.

Neuron loss occurring in neurodegenerative diseases represents just the final step in a series of events involving several cell types, other than neurons, that actively contribute to the overall pathogenic mechanisms by establishing harmful non-cell autonomous effects [...].

Targeting S100A4 with niclosamide attenuates inflammatory and profibrotic pathways in models of amyotrophic lateral sclerosis.

BACKGROUND: An increasing number of studies evidences that amyotrophic lateral sclerosis (ALS) is characterized by extensive alterations in different cell types and in different regions besides the CNS. We previously reported the upregulation in ALS models of a gene called fibroblast-specific protein-1 or S100A4, recognized as a pro-inflammatory and profibrotic factor. Since inflammation and fibrosis are often mutual-sustaining events that contribute to establish a hostile environment for organ functions, the comprehension of the elements responsible for these interconnected pathways is crucial to disclose novel aspects involved in ALS pathology. METHODS: Here, we employed fibroblasts derived from ALS patients harboring the C9orf72 hexanucleotide repeat expansion and ALS patients with no mutations in known ALS-associated genes and we downregulated S100A4 using siRNA or the S100A4 transcriptional inhibitor niclosamide. Mice overexpressing human FUS were adopted to assess the effects of niclosamide in vivo on ALS pathology. RESULTS: We demonstrated that S100A4 underlies impaired autophagy and a profibrotic phenotype, which characterize ALS fibroblasts. Indeed, its inhibition reduces inflammatory, autophagic, and profibrotic pathways in ALS fibroblasts, and interferes with different markers known as pathogenic in the disease, such as mTOR, SQSTM1/p62, STAT3, α-SMA, and NF-κB. Importantly, niclosamide in vivo treatment of ALS-FUS mice reduces the expression of S100A4, α-SMA, and PDGFRß in the spinal cord, as well as gliosis in central and peripheral nervous tissues, together with axonal impairment and displays beneficial effects on muscle atrophy, by promoting muscle regeneration and reducing fibrosis. CONCLUSION: Our findings show that S100A4 has a role in ALS-related mechanisms, and that drugs such as niclosamide which are able to target inflammatory and fibrotic pathways could represent promising pharmacological tools for ALS.

S100A4 in the Physiology and Pathology of the Central and Peripheral Nervous System.

S100A4 is a member of the large family of S100 proteins, exerting a broad range of intracellular and extracellular functions that vary upon different cellular contexts. While S100A4 has long been implicated mainly in tumorigenesis and metastatization, mounting evidence shows that S100A4 is a key player in promoting pro-inflammatory phenotypes and organ pro-fibrotic pathways in the liver, kidney, lung, heart, tendons, and synovial tissues. Regarding the nervous system, there is still limited information concerning S100A4 presence and function. It was observed that S100A4 exerts physiological roles contributing to neurogenesis, cellular motility and chemotaxis, cell differentiation, and cell-to cell communication. Furthermore, S100A4 is likely to participate to numerous pathological processes of the nervous system by affecting the functions of astrocytes, microglia, infiltrating cells and neurons and thereby modulating inflammation and immune reactions, fibrosis as well as neuronal plasticity and survival. This review summarizes the current state of knowledge concerning the localization, deregulation, and possible functions of S100A4 in the physiology of the central and peripheral nervous system. Furthermore, we highlight S100A4 as a gene involved in the pathogenesis of neurological disorders such as brain tumors, neurodegenerative diseases, and acute injuries.

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.

Fibrotic Scar in Neurodegenerative Diseases.

The process of uncontrolled internal scarring, called fibrosis, is now emerging as a pathological feature shared by both peripheral and central nervous system diseases. In the CNS, damaged neurons are not replaced by tissue regeneration, and scar-forming cells such as endothelial cells, inflammatory immune cells, stromal fibroblasts, and astrocytes can persist chronically in brain and spinal cord lesions. Although this process was extensively described in acute CNS damages, novel evidence indicates the involvement of a fibrotic reaction in chronic CNS injuries as those occurring during neurodegenerative diseases, where inflammation and fibrosis fuel degeneration. In this mini review, we discuss recent advances around the role of fibrotic scar formation and function in different neurodegenerative conditions, particularly focusing on the rising role of scarring in the pathogenesis of amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer's disease and highlighting the therapeutic relevance of targeting fibrotic scarring to slow and reverse neurodegeneration.

UsnRNP trafficking is regulated by stress granules and compromised by mutant ALS proteins.

Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic (N/C) transport and changes in alternative splicing regulation are all common traits of the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). However, whether these processes act independently from each other, or are part of a coordinated mechanism of gene expression regulation that is affected in pathogenic conditions, is still rather undefined. To answer these questions, in this work we set out to characterise the functional connections existing between ISR activation and nucleo-cytosol trafficking and nuclear localization of spliceosomal U-rich small nuclear ribonucleoproteins (UsnRNPs), the core constituents of the spliceosome, and to study how ALS-linked mutant proteins affect this interplay. Activation of the ISR induces a profound reorganization of nuclear Gems and Cajal bodies, the membrane-less particles that assist UsnRNP maturation and storage. This effect requires the cytoplasmic assembly of SGs and is associated to the disturbance of the nuclear import of UsnRNPs by the snurportin-1/importin-ß1 system. Notably, these effects are reversed by both inhibiting the ISR or upregulating importin-ß1. This indicates that SGs are major determinants of Cajal bodies assembly and that the modulation of N/C trafficking of UsnRNPs might control alternative splicing in response to stress. Importantly, the dismantling of nuclear Gems and Cajal bodies by ALS-linked mutant FUS or C9orf72-derived dipeptide repeat proteins is halted by overexpression of importin-ß1, but not by inhibition of the ISR. This suggests that changes in the nuclear localization of the UsnRNP complexes induced by mutant ALS proteins are uncoupled from ISR activation, and that defects in the N/C trafficking of UsnRNPs might play a role in ALS pathogenesis.

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-κB (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.

The S100B story: from biomarker to active factor in neural injury.

S100B is a Ca2+ -binding protein mainly concentrated in astrocytes. Its levels in biological fluids (cerebrospinal fluid, peripheral and cord blood, urine, saliva, amniotic fluid) are recognized as a reliable biomarker of active neural distress. Although the wide spectrum of diseases in which the protein is involved (acute brain injury, neurodegenerative diseases, congenital/perinatal disorders, psychiatric disorders) reduces its specificity, its levels remain an important aid in monitoring the trend of the disorder. Mounting evidence now points to S100B as a Damage-Associated Molecular Pattern molecule which, when released at high concentration, through its Receptor for Advanced Glycation Endproducts, triggers tissue reaction to damage in a series of different neural disorders. This review addresses this novel scenario, presenting data indicating that S100B levels and/or distribution in the nervous tissue of patients and/or experimental models of different neural disorders, for which the protein is used as a biomarker, are directly related to the progress of the disease: acute brain injury (ischemic/hemorrhagic stroke, traumatic injury), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis), congenital/perinatal disorders (Down syndrome, spinocerebellar ataxia-1), psychiatric disorders (schizophrenia, mood disorders), inflammatory bowel disease. In many cases, over-expression/administration of the protein induces worsening of the disease, whereas its deletion/inactivation produces amelioration. This review points out that the pivotal role of the protein resulting from these data, opens the perspective that S100B may be regarded as a therapeutic target for these different diseases, which appear to share some common features reasonably attributable to neuroinflammation, regardless their origin.

Differential toxicity of TAR DNA-binding protein 43 isoforms depends on their submitochondrial localization in neuronal cells.

TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP-43 exists as a full-length protein and as two shorter forms of 25 and 35 kDa. Full-length mutant TDP-43s found in amyotrophic lateral sclerosis patients re-localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP-43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kDa truncated form of TDP-43 is restricted to the intermembrane space, while the full-length forms also localize in the mitochondrial matrix in cultured neuronal NSC-34 cells. Interestingly, the full-length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial-transcribed mRNAs, while the 35 kDa form does not. In the light of the known differential contribution of the full-length and short isoforms to generate toxic aggregates, we propose that the presence of full-length TDP-43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP-43 forms play a major role.

Neuroinflammation in Amyotrophic Lateral Sclerosis: Role of Redox (dys)Regulation.

SIGNIFICANCE: Amyotrophic lateral sclerosis (ALS) is due to degeneration of upper and lower motor neurons in the anterior horn of the spinal cord and in the motor cortex. Mechanisms leading to motor neuron death are complex and currently the disease is untreatable. Recent Advances: Work in genetic models of ALS indicates that an imbalance in the cross talk that physiologically exists between motor neurons and the surrounding cells is eventually detrimental to motor neurons. In particular, the cascade of events collectively known as neuroinflammation and mainly characterized by a reactive phenotype of astrocytes and microglia, moderate infiltration of peripheral immune cells, and elevated levels of inflammatory mediators has been consistently observed in motor regions of the central nervous system (CNS) in sporadic and familial ALS, constituting a hallmark of the disease. Resident glial cells and infiltrated immune cells are considered among the major producers of reactive species of oxygen and nitrogen in pathological conditions of the CNS, including motor neuron diseases. CRITICAL ISSUES: The timing and exact role of oxidative stress-mediated neuroinflammation and damage to motor neurons in ALS are still not fully elucidated. FUTURE DIRECTIONS: It is clear that a major challenge in the next future will be to envisage effective strategies to modulate the neuroinflammatory response in the symptomatic stage of disease, to prevent progression of neurodegeneration through the propagation of oxidative damage. Antioxid. Redox Signal. 29, 15-36.

The Dual Role of Microglia in ALS: Mechanisms and Therapeutic Approaches.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a non-cell autonomous motor neuron loss. While it is generally believed that the disease onset takes place inside motor neurons, different cell types mediating neuroinflammatory processes are considered deeply involved in the progression of the disease. On these grounds, many treatments have been tested on ALS animals with the aim of inhibiting or reducing the pro-inflammatory action of microglia and astrocytes and counteract the progression of the disease. Unfortunately, these anti-inflammatory therapies have been only modestly successful. The non-univocal role played by microglia during stress and injuries might explain this failure. Indeed, it is now well recognized that, during ALS, microglia displays different phenotypes, from surveillant in early stages, to activated states, M1 and M2, characterized by the expression of respectively harmful and protective genes in later phases of the disease. Consistently, the inhibition of microglial function seems to be a valid strategy only if the different stages of microglia polarization are taken into account, interfering with the reactivity of microglia specifically targeting only the harmful pathways and/or potentiating the trophic ones. In this review article, we will analyze the features and timing of microglia activation in the light of M1/M2 phenotypes in the main mice models of ALS. Moreover, we will also revise the results obtained by different anti-inflammatory therapies aimed to unbalance the M1/M2 ratio, shifting it towards a protective outcome.

The Astrocytic S100B Protein with Its Receptor RAGE Is Aberrantly Expressed in SOD1G93A 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 SOD1G93A 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.