Interleukin-9 protects from microglia- and TNF-mediated synaptotoxicity in experimental multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a progressive neurodegenerative disease of the central nervous system characterized by inflammation-driven synaptic abnormalities. Interleukin-9 (IL-9) is emerging as a pleiotropic cytokine involved in MS pathophysiology. METHODS: Through biochemical, immunohistochemical, and electrophysiological experiments, we investigated the effects of both peripheral and central administration of IL-9 on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. RESULTS: We demonstrated that both systemic and local administration of IL-9 significantly improved clinical disability, reduced neuroinflammation, and mitigated synaptic damage in EAE. The results unveil an unrecognized central effect of IL-9 against microglia- and TNF-mediated neuronal excitotoxicity. Two main mechanisms emerged: first, IL-9 modulated microglial inflammatory activity by enhancing the expression of the triggering receptor expressed on myeloid cells-2 (TREM2) and reducing TNF release. Second, IL-9 suppressed neuronal TNF signaling, thereby blocking its synaptotoxic effects. CONCLUSIONS: The data presented in this work highlight IL-9 as a critical neuroprotective molecule capable of interfering with inflammatory synaptopathy in EAE. These findings open new avenues for treatments targeting the neurodegenerative damage associated with MS, as well as other inflammatory and neurodegenerative disorders of the central nervous system.

Interleukin 6 SNP rs1818879 Regulates Radiological and Inflammatory Activity in Multiple Sclerosis.

(1) Background: The clinical course of multiple sclerosis (MS) is critically influenced by the expression of different pro-inflammatory and anti-inflammatory cytokines. Interleukin 6 (IL-6) represents a major inflammatory molecule previously associated with exacerbated disease activity in relapsing remitting MS (RR-MS); however, the role of single-nucleotide polymorphisms (SNPs) in the IL-6 gene has not been fully elucidated in MS. (2) Methods: We explored in a cohort of 171 RR-MS patients, at the time of diagnosis, the associations between four IL-6 SNPs (rs1818879, rs1554606, rs1800797, and rs1474347), CSF inflammation, and clinical presentation. (3) Results: Using principal component analysis and logistic regression analysis we identified an association between rs1818879, radiological activity, and a set of cytokines, including the IL-1ß, IL-9, IL-10, and IL-13. No significant associations were found between other SNPs and clinical or inflammatory parameters. (4) Conclusions: The association between the rs1818879 polymorphism and subclinical neuroinflammatory activity suggests that interindividual differences in the IL-6 gene might influence the immune activation profile in MS.

Exercise protects from hippocampal inflammation and neurodegeneration in experimental autoimmune encephalomyelitis.

Exercise is increasingly recommended as a supportive therapy for people with Multiple Sclerosis (pwMS). While clinical research has still not disclosed the real benefits of exercise on MS disease, animal studies suggest a substantial beneficial effect on motor disability and pathological hallmarks such as central and peripheral dysregulated immune response. The hippocampus, a core area for memory formation and learning, is a brain region involved in MS pathophysiology. Human and rodent studies suggest that the hippocampus is highly sensitive to the effects of exercise, the impact of which on MS hippocampal damage is still elusive. Here we addressed the effects of chronic voluntary exercise on hippocampal function and damage in experimental autoimmune encephalomyelitis (EAE), animal model of MS. Mice were housed in standard or wheel-equipped cages starting from the day of immunization and throughout the disease course. Although running activity was reduced during the symptomatic phase, exercise significantly ameliorated motor disability. Exercise improved cognition that was assessed through the novel object recognition test and the nest building in presymptomatic and acute stages of the disease, respectively. In the acute phase exercise was shown to prevent EAE-induced synaptic plasticity abnormalities in the CA1 area, by promoting the survival of parvalbumin-positive (PV+) interneurons and by attenuating inflammation. Indeed, exercise significantly reduced microgliosis in the CA1 area, the expression of tumour necrosis factor (TNF) in microglia and, to a lesser extent, the hippocampal level of interleukin 1 beta (IL-1ß), previously shown to contribute to aberrant synaptic plasticity in the EAE hippocampus. Notably, exercise exerted a precocious and long-lasting mitigating effect on microgliosis that preceded its neuroprotective action, likely underlying the improved cognitive function observed in both presymptomatic and acute phase EAE mice. Overall, these data provide evidence that regular exercise improves cognitive function and synaptic and neuronal pathology that typically affect EAE/MS brains.

A Single Nucleotide ADA Genetic Variant Is Associated to Central Inflammation and Clinical Presentation in MS: Implications for Cladribine Treatment.

In multiple sclerosis (MS), activated T and B lymphocytes and microglial cells release various proinflammatory cytokines, promoting neuroinflammation and negatively affecting the course of the disease. The immune response homeostasis is crucially regulated by the activity of the enzyme adenosine deaminase (ADA), as evidenced in patients with genetic ADA deficiency and in those treated with cladribine tablets. We investigated in a group of patients with MS the associations of a single nucleotide polymorphism (SNP) of ADA gene with disease characteristics and cerebrospinal fluid (CSF) inflammation. The SNP rs244072 of the ADA gene was determined in 561 patients with MS. Disease characteristics were assessed at the time of diagnosis; furthermore, in 258 patients, proinflammatory and anti-inflammatory molecules were measured in the CSF. We found a significant association between rs244072 and both clinical characteristics and central inflammation. In C-carriers, significantly enhanced disability and increased CSF levels of TNF, IL-5 and RANTES was observed. In addition, lower CSF levels of the anti-inflammatory cytokine IL-10 were found. Finally, the presence of the C allele was associated with a tendency of increased lymphocyte count. In MS patients, ADA SNP rs244072 is associated with CSF inflammation and disability. The selective targeting of the ADA pathway through cladribine tablet therapy could be effective in MS by acting on a pathogenically relevant biological mechanism.

Re-Examining the Role of TNF in MS Pathogenesis and Therapy.

Multiple sclerosis (MS) is a common neurological disorder of putative autoimmune origin. Clinical and experimental studies delineate abnormal expression of specific cytokines over the course of the disease. One major cytokine that has been shown to play a pivotal role in MS is tumor necrosis factor (TNF). TNF is a pleiotropic cytokine regulating many physiological and pathological functions of both the immune system and the central nervous system (CNS). Convincing evidence from studies in human and experimental MS have demonstrated the involvement of TNF in various pathological hallmarks of MS, including immune dysregulation, demyelination, synaptopathy and neuroinflammation. However, due to the complexity of TNF signaling, which includes two-ligands (soluble and transmembrane TNF) and two receptors, namely TNF receptor type-1 (TNFR1) and type-2 (TNFR2), and due to its cell- and context-differential expression, targeting the TNF system in MS is an ongoing challenge. This review summarizes the evidence on the pathophysiological role of TNF in MS and in different MS animal models, with a special focus on pharmacological treatment aimed at controlling the dysregulated TNF signaling in this neurological disorder.

Interleukin-6 Disrupts Synaptic Plasticity and Impairs Tissue Damage Compensation in Multiple Sclerosis.

Background: Synaptic plasticity helps in reducing the clinical expression of brain damage and represents a useful mechanism to compensate the negative impact of new brain lesions in multiple sclerosis (MS). Inflammation, altering synaptic plasticity, could negatively influence the disease course in relapsing-remitting MS (RR-MS). Objective: In the present study, we explored whether interleukin (IL)-6, a major proinflammatory cytokine involved in MS pathogenesis, alters synaptic plasticity and affects the ability to compensate for ongoing brain damage. Methods: The effect of IL-6 incubation on long-term potentiation (LTP) induction was explored in vitro, in mice hippocampal slices. We also explored the correlation between the cerebrospinal fluid (CSF) levels of this cytokine and the LTP-like effect induced by the paired associative stimulation (PAS) in a group of RR-MS patients. Finally, we examined the correlation between the CSF levels of IL-6 at the time of diagnosis and the prospective disease activity in a cohort of 150 RR-MS patients. Results:In vitro LTP induction was abolished by IL-6. Consistently, in patients with MS, a negative correlation emerged between IL-6 CSF concentrations and the effect of PAS. In MS patients, longer disease duration before diagnosis was associated with higher IL-6 CSF concentrations. In addition, elevated CSF levels of IL-6 were associated with greater clinical expression of new inflammatory brain lesions, unlike in patients with low or absent IL-6 concentrations, who had a better disease course. Conclusions: IL-6 interfering with synaptic plasticity mechanisms may impair the ability to compensate the clinical manifestation of new brain lesions in RR-MS patients.

Delayed treatment of MS is associated with high CSF levels of IL-6 and IL-8 and worse future disease course.

BACKGROUND: Clinical deterioration of relapsing-remitting MS (RR-MS) patients reflects not only the number and severity of overt inflammatory and demyelinating episodes, but also subtle central damage caused by persistent exposure to inflammatory molecules. OBJECTIVE: To explore the correlation between levels of CSF inflammatory molecules at the time of diagnosis and both demographic and clinical characteristics of a large sample of RR-MS patients, as well as the predictive value of cytokine levels on their prospective disease course. METHODS: In 205 patients diagnosed with RR-MS, we measured at the time of diagnosis the CSF levels of inflammatory molecules. Clinical and MRI evaluation was collected at the time of CSF withdrawal and during a median follow-up of 3 years. RESULTS: The time interval between the first anamnestic episode of focal neurological dysfunction and RR-MS diagnosis was the main factor associated with high CSF levels of IL-6 and IL-8. Furthermore, elevated CSF levels of these cytokines correlated with enhanced risk of clinical and radiological disease reactivation, switch to second-line treatments, and with disability progression in the follow-up. CONCLUSIONS: Delayed diagnosis and treatment initiation are associated with higher CSF levels of IL-6 and IL-8 in RR-MS, leading to worsening disease course and poor response to treatments.

Tumor Necrosis Factor and Interleukin-1ß Modulate Synaptic Plasticity during Neuroinflammation.

Cytokines are constitutively released in the healthy brain by resident myeloid cells to keep proper synaptic plasticity, either in the form of Hebbian synaptic plasticity or of homeostatic plasticity. However, when cytokines dramatically increase, establishing a status of neuroinflammation, the synaptic action of such molecules remarkably interferes with brain circuits of learning and cognition and contributes to excitotoxicity and neurodegeneration. Among others, interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF) are the best studied proinflammatory cytokines in both physiological and pathological conditions and have been invariably associated with long-term potentiation (LTP) (Hebbian synaptic plasticity) and synaptic scaling (homeostatic plasticity), respectively. Multiple sclerosis (MS) is the prototypical neuroinflammatory disease, in which inflammation triggers excitotoxic mechanisms contributing to neurodegeneration. IL-ß and TNF are increased in the brain of MS patients and contribute to induce the changes in synaptic plasticity occurring in MS patients and its animal model, the experimental autoimmune encephalomyelitis (EAE). This review will introduce and discuss current evidence of the role of IL-1ß and TNF in the regulation of synaptic strength at both physiological and pathological levels, in particular speculating on their involvement in the synaptic plasticity changes observed in the EAE brain.

miR-135a Regulates Synaptic Transmission and Anxiety-Like Behavior in Amygdala.

MicroRNAs are a class of non-coding RNAs with a growing relevance in the regulation of gene expression related to brain function and plasticity. They have the potential to orchestrate complex phenomena, such as the neuronal response to homeostatic challenges. We previously demonstrated the involvement of miR-135a in the regulation of early stress response. In the present study, we examine the role of miR-135a in stress-related behavior. We show that the knockdown (KD) of miR-135a in the mouse amygdala induces an increase in anxiety-like behavior. Consistently with behavioral studies, electrophysiological experiments in acute brain slices indicate an increase of amygdala spontaneous excitatory postsynaptic currents, as a result of miR-135a KD. Furthermore, we presented direct evidences, by in vitro assays and in vivo miRNA overexpression in the amygdala, that two key regulators of synaptic vesicle fusion, complexin-1 and complexin-2, are direct targets of miR-135a. In vitro analysis of miniature excitatory postsynaptic currents on miR-135a KD primary neurons indicates unpaired quantal excitatory neurotransmission. Finally, increased levels of complexin-1 and complexin-2 proteins were detected in the mouse amygdala after acute stress, accordingly to the previously observed stress-induced miR-135a downregulation. Overall, our results unravel a previously unknown miRNA-dependent mechanism in the amygdala for regulating anxiety-like behavior, providing evidences of a physiological role of miR-135a in the modulation of presynaptic mechanisms of glutamatergic neurotransmission.

RNA-binding protein HuR and the members of the miR-200 family play an unconventional role in the regulation of c-Jun mRNA.

Post-transcriptional gene regulation is a fundamental step for coordinating cellular response in a variety of processes. RNA-binding proteins (RBPs) and microRNAs (miRNAs) are the most important factors responsible for this regulation. Here we report that different components of the miR-200 family are involved in c-Jun mRNA regulation with the opposite effect. While miR-200b inhibits c-Jun protein production, miR-200a tends to increase the JUN amount through a stabilization of its mRNA. This action is dependent on the presence of the RBP HuR that binds the 3'UTR of c-Jun mRNA in a region including the mir-200a binding site. The position of the binding site is fundamental; by mutating this site, we demonstrate that the effect is not micro-RNA specific. These results indicate that miR-200a triggers a microRNA-mediated stabilization of c-Jun mRNA, promoting the binding of HuR with c-Jun mRNA. This is the first example of a positive regulation exerted by a microRNA on an important oncogene in proliferating cells.

Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

miR-206, a member of the so-called myomiR family, is largely acknowledged as a specific, positive regulator of skeletal muscle differentiation. A growing body of evidence also suggests a tumor suppressor function for miR-206, as it is frequently downregulated in various types of cancers. In this study, we show that miR-206 directly targets cyclin D1 and contributes to the regulation of CCND1 gene expression in both myogenic and non-muscle, transformed cells. We demonstrate that miR-206, either exogenous or endogenous, reduces cyclin D1 levels and proliferation rate in C2C12 cells without promoting differentiation, and that miR-206 knockdown in terminally differentiated C2C12 cells leads to cyclin D1 accumulation in myotubes, indicating that miR-206 might be involved in the maintenance of the post-mitotic state. Targeting of cyclin D1 might also account, at least in part, for the tumor-suppressor activity suggested for miR-206 in previous studies. Accordingly, the analysis of neoplastic and matched normal lung tissues reveals that miR-206 downregulation in lung tumors correlates, in most cases, with higher cyclin D1 levels. Moreover, gain-of-function experiments with cancer-derived cell lines and with in vitro transformed cells indicate that miR-206-mediated cyclin D1 repression is directly coupled to growth inhibition. Altogether, our data highlight a novel activity for miR-206 in skeletal muscle differentiation and identify cyclin D1 as a major target that further strengthens the tumor suppressor function proposed for miR-206.