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 [...].

The Histamine and Multiple Sclerosis Alliance: Pleiotropic Actions and Functional Validation.

Multiple sclerosis (MS) is a disease with a resilient inflammatory component caused by accumulation into the CNS of inflammatory infiltrates and macrophage/microglia contributing to severe demyelination and neurodegeneration. While the causes are still in part unclear, key pathogenic mechanisms are the direct loss of myelin-producing cells and/or their impairment caused by the immune system. Proposed etiology includes genetic and environmental factors triggered by viral infections. Although several diagnostic methods and new treatments are under development, there is no curative but only palliative care against the relapsing-remitting or progressive forms of MS. In recent times, there has been a boost of awareness on the role of histamine signaling in physiological and pathological functions of the nervous system. Particularly in MS, evidence is raising that histamine might be directly implicated in the disease by acting at different cellular and molecular levels. For instance, constitutively active histamine regulates the differentiation of oligodendrocyte precursors, thus playing a central role in the remyelination process; histamine reduces the ability of myelin-autoreactive T cells to adhere to inflamed brain vessels, a crucial step in the development of MS; histamine levels are found increased in the cerebrospinal fluid of MS patients. The aim of the present work is to present further proofs about the alliance of histamine with MS and to introduce the most recent and innovative histamine paradigms for therapy. We will report on how a long-standing molecule with previously recognized immunomodulatory and neuroprotective functions, histamine, might still provide a renewed and far-reaching role in MS.

Novel P2X7 Antagonist Ameliorates the Early Phase of ALS Disease and Decreases Inflammation and Autophagy in SOD1-G93A Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a disease with a resilient neuroinflammatory component caused by activated microglia and infiltrated immune cells. How to successfully balance neuroprotective versus neurotoxic actions through the use of anti-inflammatory agents is still under debate. There has been a boost of awareness regarding the role of extracellular ATP and purinergic receptors in modulating the physiological and pathological mechanisms in the nervous system. Particularly in ALS, it is known that the purinergic ionotropic P2X7 receptor plays a dual role in disease progression by acting at different cellular and molecular levels. In this context, we previously demonstrated that the P2X7 receptor antagonist, brilliant blue G, reduces neuroinflammation and ameliorates some of the pathological features of ALS in the SOD1-G93A mouse model. Here, we test the novel, noncommercially available, and centrally permeant Axxam proprietary P2X7 antagonist, AXX71, in SOD1-G93A mice, by assessing some behavioral and molecular parameters, among which are disease progression, survival, gliosis, and motor neuron wealth. We demonstrate that AXX71 affects the early symptomatic phase of the disease by reducing microglia-related proinflammatory markers and autophagy without affecting the anti-inflammatory markers or motor neuron survival. Our results suggest that P2X7 modulation can be further investigated as a therapeutic strategy in preclinical studies, and exploited in ALS clinical trials.

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.

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.

Omics-based exploration and functional validation of neurotrophic factors and histamine as therapeutic targets in ALS.

A plethora of genetic and molecular mechanisms have been implicated in the pathophysiology of the heterogeneous and multifactorial amyotrophic lateral sclerosis (ALS) disease, and hence the conventional "one target-one drug" paradigm has failed so far to provide effective therapeutic solutions, precisely because of the complex nature of ALS. This review intends to highlight how the integration of emerging "omics" approaches may provide a rational foundation for the comprehensive exploration of molecular pathways and dynamic interactions involved in ALS, for the identification of candidate targets and biomarkers that will assist in the rapid diagnosis and prognosis, lastly for the stratification of patients into different subgroups with the aim of personalized therapeutic strategies. To this purpose, particular emphasis will be placed on some potential therapeutic targets, including neurotrophic factors and histamine signaling that both have emerged as dysregulated at different omics levels in specific subgroups of ALS patients, and have already shown promising results in in vitro and in vivo models of ALS. To conclude, we will discuss about the utility of using integrated omics coupled with network-based approaches to provide additional guidance for personalization of medicine applications in ALS.

P2X7 activation enhances skeletal muscle metabolism and regeneration in SOD1G93A mouse model of amyotrophic lateral sclerosis.

Muscle weakness plays an important role in neuromuscular disorders comprising amyotrophic lateral sclerosis (ALS). However, it is not established whether muscle denervation originates from the motor neurons, the muscles or more likely both. Previous studies have shown that the expression of the SOD1G93A mutation in skeletal muscles causes denervation of the neuromuscular junctions, inability to regenerate and consequent atrophy, all clear symptoms of ALS. In this work, we used SOD1G93A mice, a model that best mimics some pathological features of both familial and sporadic ALS, and we investigated some biological effects induced by the activation of the P2X7 receptor in the skeletal muscles. The P2X7, belonging to the ionotropic family of purinergic receptors for extracellular ATP, is abundantly expressed in the healthy skeletal muscles, where it controls cell duplication, differentiation, regeneration or death. In particular, we evaluated whether an in vivo treatment in SOD1G93A mice with the P2X7 specific agonist 2'(3')-O-(4-Benzoylbenzoyl) adenosine5'-triphosphate (BzATP) just before the onset of a pathological neuromuscular phenotype could exert beneficial effects in the skeletal muscles. Our findings indicate that stimulation of P2X7 improves the innervation and metabolism of myofibers, moreover elicits the proliferation/differentiation of satellite cells, thus preventing the denervation atrophy of skeletal muscles in SOD1G93A mice. Overall, this study suggests that a P2X7-targeted and site-specific modulation might be a strategy to interfere with the complex multifactorial and multisystem nature of ALS.

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.

Stimulation of P2X7 Enhances Whole Body Energy Metabolism in Mice.

The P2X7 receptor, a member of the ionotropic purinergic P2X family of extracellular ATP-gated receptors, exerts strong trophic effects when tonically activated in cells, in addition to cytotoxic effects after a sustained activation. Because of its widespread distribution, P2X7 regulates several cell- and tissue-specific physiological functions, and is involved in a number of disease conditions. A novel role has recently emerged for P2X7 in the regulation of glucose and energy metabolism. In previous work, we have demonstrated that genetic depletion, and to a lesser extent also pharmacological inhibition of P2X7, elicits a significant decrease of the whole body energy expenditure and an increase of the respiratory exchange ratio. In the present work, we have investigated the effects of P2X7 stimulation in vivo on the whole body energy metabolism. Adult mice were daily injected with the specific P2X7 agonist 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate for 1 week and subjected to indirect calorimetric analysis for 48 h. We report that 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate increases metabolic rate and O2 consumption, concomitantly decreasing respiratory rate and upregulating NADPH oxidase 2 in gastrocnemius and tibialis anterior muscles. Our results indicate a major impact on energy homeostasis and muscle metabolism by activation of P2X7.

Histamine Is an Inducer of the Heat Shock Response in SOD1-G93A Models of ALS.

(1) Background: Amyotrophic lateral sclerosis (ALS) is a multifactorial non-cell autonomous disease where activation of microglia and astrocytes largely contributes to motor neurons death. Heat shock proteins have been demonstrated to promote neuronal survival and exert a strong anti-inflammatory action in glia. Having previously shown that the pharmacological increase of the histamine content in the central nervous system (CNS) of SOD1-G93A mice decreases neuroinflammation, reduces motor neuron death, and increases mice life span, here we examined whether this effect could be mediated by an enhancement of the heat shock response. (2) Methods: Heat shock protein expression was analyzed in vitro and in vivo. Histamine was provided to primary microglia and NSC-34 motor neurons expressing the SOD1-G93A mutation. The brain permeable histamine precursor histidine was chronically administered to symptomatic SOD1-G93A mice. Spine density was measured by Golgi-staining in motor cortex of histidine-treated SOD1-G93A mice. (3) Results: We demonstrate that histamine activates the heat shock response in cultured SOD1-G93A microglia and motor neurons. In SOD1-G93A mice, histidine augments the protein content of GRP78 and Hsp70 in spinal cord and cortex, where the treatment also rescues type I motor neuron dendritic spine loss. (4) Conclusion: Besides the established histaminergic neuroprotective and anti-inflammatory effects, the induction of the heat shock response in the SOD1-G93A model by histamine confirms the importance of this pathway in the search for successful therapeutic solutions to treat ALS.