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Clinical and animal model studies have implicated inflammation and glial and peripheral immune cell responses in the pathophysiology of spinal cord injury (SCI). A key player in the inflammatory response after SCI is the pleiotropic cytokine tumor necrosis factor (TNF), which exists both in both a transmembrane (tmTNF) and a soluble (solTNF) form. In the present study, we extend our previous findings of a therapeutic effect of topically blocking solTNF signaling after SCI for three consecutive days on lesion size and functional outcome to study the effect on spatio-temporal changes in the inflammatory response after SCI in mice treated with the selective solTNF inhibitor XPro1595 and compared to saline-treated mice. We found that despite comparable TNF and TNF receptor levels between XPro1595- and saline-treated mice, XPro1595 transiently decreased pro-inflammatory interleukin (IL)-1β and IL-6 levels and increased pro-regenerative IL-10 levels in the acute phase after SCI. This was complemented by a decrease in the number of infiltrated leukocytes (macrophages and neutrophils) in the lesioned area of the spinal cord and an increase in the number of microglia in the peri-lesion area 14 days after SCI, followed by a decrease in microglial activation in the peri-lesion area 21 days after SCI. This translated into increased myelin preservation and improved functional outcomes in XPro1595-treated mice 35 days after SCI. Collectively, our data suggest that selective targeting of solTNF time-dependently modulates the neuroinflammatory response by favoring a pro-regenerative environment in the lesioned spinal cord, leading to improved functional outcomes.

We have shown that KRAS–TP53 genomic coalteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS–TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-KrasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms the trafficking and functional dynamics of neutrophils to overcome T-cell exclusion and controls tumor growth in a T cell–dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembrane TNF–TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell–neutrophil cross-talk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC.Significance:By decoding connections between high-risk tumor genotypes, cell-autonomous inflammatory programs, and myeloid-enriched/T cell–excluded contexts, we identify a novel role for neutrophil-derived TNF in sustaining immunosuppression and stromal inflammation in pancreatic tumor microenvironments. This work offers a conceptual framework by which targeting context-dependent TNF signaling may overcome hallmarks of chemoresistance in pancreatic cancer.

Differentially expressed pathways comparing transcriptomes in KRAS-TP53 co-altered (n=23) and KRAS-altered/TP53WT (n=5) derived from Cancer Cell Line Encyclopedia

Activation of the ER stress sensor IRE1α contributes to neuronal development and is known to induce neuronal remodelingin vitroandin vivo. On the other hand, excessive IRE1 activity is often detrimental and may contribute to neurodegeneration. To determine the consequences of increased activation of IRE1α, we used a mouse model expressing a C148S variant of IRE1α with increased and sustained activation. Surprisingly, the mutation did not affect the differentiation of highly secretory antibody-producing cells, but exhibited a strong protective effect in a mouse model of experimental autoimmune encephalomyelitis (EAE). Significant improvement in motor function was found in IRE1C148S mice with EAE relative to WT mice. Coincident with this improvement, there was reduced microgliosis in the spinal cord of IRE1C148S mice, with reduced expression of pro-inflammatory cytokine genes. This was accompanied by reduced axonal degeneration and enhanced CNPase levels, suggestiing improved myelin integrity. Interestingly, while the IRE1C148S mutation is expressed in all cells, the reduction in proinflammatory cytokines and in the activation of microglial activation marker IBA1, along with preservation of phagocytic gene expression, all point to microglia as the cell type contributing to the clinical improvement in IRE1C148S animals. Our data suggest that sustained increase in IRE1α activity can be protectivein vivo, and that this protection is cell type and context dependent. Considering the overwhelming but conflicting evidence for the role of the ER stress in neurological diseases, a better understanding of the function of ER stress sensors in physiological contexts is clearly needed.

Pre-clinical studies place tumor necrosis factor (TNF) as a central player in the inflammatory response after spinal cord injury (SCI), and blocking its production and/or activity has been proposed as a possible treatment option after SCI. This systematic review provides an overview of the literature on the temporal and cellular expression of TNF after SCI and clarifies the potential for its therapeutic manipulation in SCI. A systematic search was performed in EMBASE (Ovid), MEDLINE (Ovid), and Web of Science (Core Collection). The search terms were the MeSH forms of tumor necrosis factor and spinal cord injury in the different databases, and the last search was performed on February 3, 2021. We found twenty-four articles examining the expression of TNF, with most using a thoracic contusive SCI model in rodents. Two articles described the expression of TNF receptors in the acute phase after SCI. Twenty-one articles described the manipulation of TNF signaling using genetic knock-out, pharmaceutical inhibition, or gain-of-function approaches. Overall, TNF expression increased rapidly after SCI, within the first hours, in resident cells (neurons, astrocytes, oligodendrocytes, and microglia) and again in macrophages in the chronic phase after injury. The review underscores the complexity of TNF's role after SCI and indicates that TNF inhibition is a promising therapeutic option. This review concludes that TNF plays a significant role in the inflammatory response after SCI and suggests that targeting TNF signaling is a feasible therapeutic approach.

BackgroundThe inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) is involved in immune signaling by bridging the interactions between inflammasome sensors and caspase-1. Strong experimental evidence has shown that ASC−/−mice are protected from disease progression in animal models of multiple sclerosis (MS), suggesting that targeting inflammasome activation via ASC inhibition may be a promising therapeutic strategy in MS. Thus, the goal of our study is to test the efficacy of IC100, a novel humanized antibody targeting ASC, in preventing and/or suppressing disease in the experimental autoimmune encephalomyelitis (EAE) model of MS.MethodsWe employed the EAE model of MS where disease was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55). Mice were treated with vehicle or increasing doses of IC100 (10, 30, and 45 mg/kg) and clinical disease course was evaluated up to 35 days post EAE induction. Immune cell infiltration into the spinal cord and microglia responses were assessed.ResultsWe show that IC100 treatment reduced the severity of EAE when compared to vehicle-treated controls. At a dose of 30 mg/kg, IC100 significantly reduced the number of CD4+and CD8+T cells and CD11b+MHCII+activated myeloid cells entering the spinal cord from the periphery, and reduced the number of total and activated microglia.ConclusionsThese data indicate that IC100 suppresses the immune-inflammatory response that drives EAE development and progression, thereby identifying ASC as a promising target for the treatment of MS as well as other neurological diseases with a neuroinflammatory component.

The intestinal milieu harbours the gut microbiota, consisting of a complex community of bacteria, archaea, fungi, viruses and protozoans that bring to the host organism an endowment of cells and genes more numerous than its own. In the last 10 years, mounting evidence has highlighted the prominent influence of the gut mutualistic bacterial communities on human health. Microbial colonization occurs alongside with immune system development and plays a role in intestinal physiology. The community of the gut microbiota does not undergo significant fluctuations throughout adult life. However, bacterial infections, antibiotic treatment, lifestyle, surgery and diet might profoundly affect it. Gut microbiota dysbiosis, defined as marked alterations in the amount and function of the intestinal microorganisms, is correlated with the aetiology of chronic noncommunicable diseases, ranging from cardiovascular, neurologic, respiratory and metabolic illnesses to cancer. In this review, we focus on the interplay among gut microbiota, diet and host to provide a perspective on the role of microbiota and their unique metabolites in the pathogenesis and/or progression of various human disorders. We discuss interventions based on microbiome studies, that is faecal microbiota transplantation, probiotics and prebiotics, to introduce the concept that correcting gut dysbiosis can ameliorate disease symptoms, thus offering a new approach towards disease treatment.

Background: For decades, mitochondrial dysfunctions and the generation of reactive oxygen species have been proposed to promote the development and progression of the amyloid pathology in Alzheimer’s disease, but this association is still debated. In particular, it is still unclear if mitochondrial dysfunctions are a trigger or rather a consequence of the formation of amyloid aggregates, and in particular, the role of the different mitochondrial oxidative phosphorylation complexes in Alzheimer’s patients’ brain remains poorly understood. Methods: To study how mitochondrial Complex III defects affect amyloid beta pathology in vivo , we partially knocked out mitochondrial Complex III (CIII KO ) in mature forebrain neurons of an Alzheimer’s mouse model that develops plaque pathology (APP/PS1). Results: We found that Complex III dysfunction in adult neurons induced mild oxidative stress which did not correlate with increased amyloid beta accumulation. In fact, CIII KO -AD mice showed decreased plaque number, decreased Aβ42 toxic fragment and altered amyloid precursor protein cleavage pathway. Conclusions: Our results support a model in which mitochondrial dysfunction is not the cause of amyloid oligomer accumulation but rather a consequence of amyloid beta toxicity.

Targeting the protein–protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) is a potential therapeutic strategy to control diseases involving oxidative stress. Here, six classes of known small-molecule Keap1–Nrf2 PPI inhibitors were dissected into 77 fragments in a fragment-based deconstruction reconstruction (FBDR) study and tested in four orthogonal assays. This gave 17 fragment hits of which six were shown by X-ray crystallography to bind in the Keap1 Kelch binding pocket. Two hits were merged into compound 8 with a 220–380-fold stronger affinity (Ki = 16 μM) relative to the parent fragments. Systematic optimization resulted in several novel analogues with Ki values of 0.04–0.5 μM, binding modes determined by X-ray crystallography, and enhanced microsomal stability. This demonstrates how FBDR can be used to find new fragment hits, elucidate important ligand–protein interactions, and identify new potent inhibitors of the Keap1–Nrf2 PPI. Targeting the protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) is a potential therapeutic strategy to control diseases involving oxidative stress. Here, six classes of known small-molecule Keap1-Nrf2 PPI inhibitors were dissected into 77 fragments in a fragment-based deconstruction reconstruction (FBDR) study and tested in four orthogonal assays. This gave 17 fragment hits of which six were shown by X-ray crystallography to bind in the Keap1 Kelch binding pocket. Two hits were merged into compound 8 with a 220-380-fold stronger affinity (Ki = 16 μM) relative to the parent fragments. Systematic optimization resulted in several novel analogues with Ki values of 0.04-0.5 μM, binding modes determined by X-ray crystallography, and enhanced microsomal stability. This demonstrates how FBDR can be used to find new fragment hits, elucidate important ligand-protein interactions, and identify new potent inhibitors of the Keap1-Nrf2 PPI.

Spinal cord injury (SCI) is a devastating condition consisting of an instant primary mechanical injury followed by a secondary injury that progresses for weeks to months. The cytokine tumor necrosis factor (TNF) plays an important role in the pathophysiology of SCI. We investigated the effect of myeloid TNF ablation (peripheral myeloid cells (macrophages and neutrophils) and microglia) versus central myeloid TNF ablation (microglia) in a SCI contusion model. We show that TNF ablation in macrophages and neutrophils leads to reduced lesion volume and improved functional outcome after SCI. In contrast, TNF ablation in microglia only or TNF deficiency in all cells had no effect. TNF levels tended to be decreased 3 h post-SCI in mice with peripheral myeloid TNF ablation and was significantly decreased 3 days after SCI. Leukocyte and microglia populations and all other cytokines (IL-1&beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IFN&gamma, ) and chemokines (CCL2, CCL5, and CXCL1) investigated, in addition to TNFR1 and TNFR2, were comparable between genotypes. Analysis of post-SCI signaling cascades demonstrated that the MAPK kinase SAPK/JNK decreased and neuronal Bcl-XL levels increased post-SCI in mice with ablation of TNF in peripheral myeloid cells. These findings demonstrate that peripheral myeloid cell-derived TNF is pathogenic in SCI.

Purpose To determine the effectiveness of etanercept, a tumor necrosis factor (TNF) inhibitor, in conferring neuroprotection to retinal ganglion cells (RGCs) and improving visual outcomes after optic nerve trauma with either optic nerve crush (ONC) or sonication-induced traumatic optic neuropathy (SI-TON) in mice. Methods Mouse optic nerves were unilaterally subjected to ONC (n = 20) or SI-TON (n = 20). TNF expression was evaluated by using immunohistochemistry and quantitative RT-PCR (qRT-PCR) in optic nerves harvested 6 and 24 hours post ONC (n = 10) and SI-TON (n = 10). Mice in each injury group received daily subcutaneous injections of either etanercept (10 mg/kg of body weight; five mice) or vehicle (five mice) for 7 days. Pattern electroretinograms were performed on all mice at 1 and 2 weeks after injury. ONC mice were killed at 2 weeks after injury, while SI-TON mice were euthanized at 4 weeks after injury. Whole retina flat-mounts were used for RGC quantification. Results Immunohistochemistry and qRT-PCR showed upregulation of TNF protein and gene expression within 24 hours after injury. In both models, etanercept use immediately following optic nerve injury led to higher RGC survival when compared to controls, which was comparable between the two models (24.23% in ONC versus 20.42% in SI-TON). In both models, 1 and 2 weeks post injury, mice treated with etanercept had significantly higher a-wave amplitudes than untreated injured controls. Conclusions Treatment with etanercept significantly reduced retinal damage and improved visual function in both animal models of TON. These findings suggest that reducing TNF activity in injured optic nerves constitutes an effective therapeutic approach in an acute setting.

The pleotropic cytokine tumor necrosis factor (TNF) is involved in the pathophysiology of multiple sclerosis (MS). In various models of MS, including experimental autoimmune encephalomyelitis (EAE), the membrane-bound form of TNF (tmTNF), which signals primarily via TNFR2, mediates protective and reparative effects, whereas the soluble form (solTNF), which signals primarily via TNFR1, promotes pro-inflammatory and detrimental functions. In this study, we investigated the role of TNFR2 expressed in oligodendrocytes in the early phase of EAE pathogenesis. We demonstrated that mice with specific ablation of oligodendroglial TNFR2 displayed early onset and higher peak of motor dysfunction when subjected to EAE, in advance of which accelerated infiltration of immune cells was observed as early as 10 days post EAE induction. The immune cell influx was preceded by microglial activation and increased blood brain barrier permeability. Lack of oligodendroglial TNFR2 accelerated the expression of inflammatory cytokines as well as expression and activation of the inflammasome. Gene expression profiling of oligodendrocytes sorted from the spinal cord 14 days post EAE induction showed robust upregulation of inflammatory genes, some of which were elevated in cells lacking TNFR2 compared to controls. Together, our data demonstrate that oligodendrocytes are directly involved in inflammation and immune modulation in CNS disease and this function is regulated, at least in part, by TNFR2.

Background: Although tumor necrosis factor (TNF) inhibitors are used to treat chronic inflammatory diseases, there is little information about how long-term inhibition of TNF affects the homeostatic functions that TNF maintains in the intact CNS. Materials and methods: To assess whether developmental TNF deficiency causes alterations in the naïve CNS, we estimated the number of proliferating cells, microglia, and neurons in the developing neocortex of E13.5, P7 and adult TNF knock out (TNF −/−) mice and wildtype (WT) littermates. We also measured changes in gene and protein expression and monoamine levels in adult WT and TNF −/− mice. To evaluate long-term effects of TNF inhibitors, we treated healthy adult C57BL/6 mice with either saline, the selective soluble TNF inhibitor XPro1595, or the nonselective TNF inhibitor etanercept. We estimated changes in cell number and protein expression after two months of treatment. We assessed the effects of TNF deficiency on cognition by testing adult WT and TNF −/− mice and mice treated with saline, XPro1595, or etanercept with specific behavioral tasks. Results: TNF deficiency decreased the number of proliferating cells and microglia and increased the number of neurons. At the same time, TNF deficiency decreased the expression of WNT signaling-related proteins, specifically Collagen Triple Helix Repeat Containing 1 (CTHRC1) and Frizzled receptor 6 (FZD6). In contrast to XPro1595, long-term inhibition of TNF with etanercept in adult C57BL/6 mice decreased the number of BrdU + cells in the granule cell layer of the dentate gyrus. Etanercept, but not XPro1595, also impaired spatial learning and memory in the Barnes maze memory test. Conclusion: TNF deficiency impacts the organization of neurogenic zones and alters the cell composition in brain. Long-term inhibition of TNF with the nonselective TNF inhibitor etanercept, but not the soluble TNF inhibitor XPro1595, decreases neurogenesis in the adult mouse hippocampus and impairs learning and memory after two months of treatment.

Neuroinflammation is the coordinated response of the central nervous system (CNS) to threats to its integrity posed by a variety of conditions, including autoimmunity, pathogens and trauma. Activated astrocytes, in concert with other cellular elements of the CNS and immune system, are important players in the modulation of the neuroinflammatory response. During neurological disease, they produce and respond to cellular signals that often lead to dichotomous processes, which can promote further damage or contribute to repair. This occurs also in multiple sclerosis (MS), where astrocytes are now recognized as key components of its immunopathology. Evidence supporting this role has emerged not only from studies in MS patients, but also from animal models, among which the experimental autoimmune encephalomyelitis (EAE) model has proved especially instrumental. Based on this premise, the purpose of the present review is to summarize the current knowledge of astrocyte behavior in MS and EAE. Following a brief description of the pathological characteristics of the two diseases and the main functional roles of astrocytes in CNS physiology, we will delve into the specific responses of this cell population, analyzing MS and EAE in parallel. We will define the temporal and anatomical profile of astroglial activation, then focus on key processes they participate in. These include: (1) production and response to soluble mediators (e.g., cytokines and chemokines), (2) regulation of oxidative stress, and (3) maintenance of BBB integrity and function. Finally, we will review the state of the art on the available methods to measure astroglial activation in vivo in MS patients, and how this could be exploited to optimize diagnosis, prognosis and treatment decisions. Ultimately, we believe that integrating the knowledge obtained from studies in MS and EAE may help not only better understand the pathophysiology of MS, but also uncover new signals to be targeted for therapeutic intervention.

Tumor necrosis factor (TNF) is associated with the pathophysiology of various neurological disorders, including multiple sclerosis. It exists as a transmembrane form tmTNF, signaling via TNF receptor 2 (TNFR2) and TNFR1, and a soluble form, solTNF, signaling via TNFR1. Multiple sclerosis is associated with the detrimental effects of solTNF acting through TNFR1, while tmTNF promotes repair and remyelination. Here we demonstrate that oligodendroglial TNFR2 is a key mediator of tmTNF-dependent protection in experimental autoimmune encephalomyelitis (EAE). CNP-cre:TNFR2fl/flmice with TNFR2 ablation in oligodendrocytes show exacerbation of the disease with increased axon and myelin pathology, reduced remyelination, and increased loss of oligodendrocyte precursor cells and mature oligodendrocytes. The clinical course of EAE is not improved by the solTNF inhibitor XPro1595 in CNP-cre:TNFR2fl/flmice, indicating that for tmTNF to promote recovery TNFR2 in oligodendrocytes is required. We show that TNFR2 drives differentiation of oligodendrocyte precursor cells, but not proliferation or survival. TNFR2 ablation leads to dysregulated expression of microRNAs, among which are regulators of oligodendrocyte differentiation and inflammation, including miR-7a. Our data provide the first directin vivoevidence that TNFR2 in oligodendrocytes is important for oligodendrocyte differentiation, thereby sustaining tmTNF-dependent repair in neuroimmune disease. Our studies identify TNFR2 in the CNS as a molecular target for the development of remyelinating agents, addressing the most pressing need in multiple sclerosis therapy nowadays.SIGNIFICANCE STATEMENTOur study, using novel TNF receptor 2 (TNFR2) conditional KO mice with selective TNFR2 ablation in oligodendrocytes, provides the first direct evidence that TNFR2 is an important signal for oligodendrocyte differentiation. Following activation by transmembrane TNF, TNFR2 initiates pathways that drive oligodendrocytes into a reparative mode contributing to remyelination following disease. This identifies TNFR2 as a new molecular target for the development of therapeutic agents in multiple sclerosis.

Background Phagocytosis is essential for maintenance of normal homeostasis and healthy tissue. As such, it is a therapeutic target for a wide range of clinical applications. The development of phenotypic screens targeting phagocytosis has lagged behind, however, due to the difficulties associated with image-based quantification of phagocytic activity. New method We present a robust algorithm and cell-based assay system for high content analysis of phagocytic activity. The method utilizes fluorescently labeled beads as a phagocytic substrate with defined physical properties. The algorithm employs statistical modeling to determine the mean fluorescence of individual beads within each image, and uses the information to conduct an accurate count of phagocytosed beads. In addition, the algorithm conducts detailed and sophisticated analysis of cellular morphology, making it a standalone tool for high content screening. Results We tested our assay system using microglial cultures. Our results recapitulated previous findings on the effects of microglial stimulation on cell morphology and phagocytic activity. Moreover, our cell-level analysis revealed that the two phenotypes associated with microglial activation, specifically cell body hypertrophy and increased phagocytic activity, are not highly correlated. This novel finding suggests the two phenotypes may be under the control of distinct signaling pathways. Comparison with existing methods We demonstrate that our assay system outperforms preexisting methods for quantifying phagocytic activity in multiple dimensions including speed, accuracy, and resolution. Conclusions We provide a framework to facilitate the development of high content assays suitable for drug screening. For convenience, we implemented our algorithm in a standalone software package, PuntoMorph.

Background: This multicenter, prospective study evaluated the determinants of zero-fluoroscopy (ZFL) ablation of supraventricular tachycardias. Methods and Results: Four hundred thirty patients (215 male, 55.4±22.1 years) with indication to electrophysiological study or ablation of supraventricular tachycardias were enrolled. All participating physicians agreed to follow the as low as reasonably achievable policy. A procedure was defined as ZFL when no fluoroscopy was used. The total fluoroscopy time inversely correlated to the number of procedures previously performed by each operator since study start ( r =−0.112; P =0.02). Two hundred eighty-nine procedures (67.2%) were ZFL; multivariable analysis identified as predictors of ZFL: procedure after the 30th for each operator, compared with procedures up to the ninth ( P =0.011; hazard ratio, 3.49; 95% confidence interval [CI], 1.79–6.80); the type of arrhythmia ( P =0.031; electrophysiological study and atrioventricular nodal reentry tachycardia ablation having the highest probability of ZFL; hazard ratio, 6.87; 95% CI, 2.08–22.7 and hazard ratio, 2.02; 95% CI, 1.04–3.91, respectively); the operator’s ( P =0.002) and patient’s age ( P =0.009). Among operators, achievement of ZFL varied from 0% to 100%; 8 (22.8%) operators achieved ZFL in 75% of their procedures. The probability of ZFL increased by 2.8% (hazard ratio, 0.98; 95% CI, 0.97–0.99) as patient’s age decreased by 1 year. Acute procedural success was obtained in all cases. Conclusions: The use of 3-dimensional mapping system completely avoided the use of fluoroscopy in most cases, with very low fluoroscopy time in the remaining and high safety and effectiveness profiles. Achievement of ZFL was predicted by the type of arrhythmia, operator’s experience, and patient’s age.

Mitochondrial dysfunction has been implicated in the pathophysiology of neurodegenerative disorders, including multiple sclerosis (MS). To date, the investigation of mitochondrial dysfunction in MS has focused exclusively on neurons, with no studies exploring whether dysregulation of mitochondrial bioenergetics and/or genetics in oligodendrocytes might be associated with the etiopathogenesis of MS and other demyelinating syndromes. To address this question, we established a mouse model where mitochondrial DNA (mtDNA) double-strand breaks (DSBs) were specifically induced in myelinating oligodendrocytes (PLP:mtPstI mice) by expressing a mitochondrial-targeted endonuclease, mtPstI, starting at 3 weeks of age. In both female and male mice, DSBs of oligodendroglial mtDNA caused impairment of locomotor function, chronic demyelination, glial activation, and axonal degeneration, which became more severe with time of induction. In addition, after short transient induction of mtDNA DSBs, PLP:mtPstI mice showed an exacerbated response to experimental autoimmune encephalomyelitis. Together, our data demonstrate that mtDNA damage can cause primary oligodendropathy, which in turn triggers demyelination, proving PLP:mtPstI mice to be a useful tool to study the pathological consequences of mitochondrial dysfunction in oligodendrocytes. In addition, the demyelination and axonal loss displayed by PLP:mtPstI mice recapitulate some of the key features of chronic demyelinating syndromes, including progressive MS forms, which are not accurately reproduced in the models currently available. For this reason, the PLP:mtPstI mouse represents a unique and much needed platform for testing remyelinating therapies.SIGNIFICANCE STATEMENTIn this study, we show that oligodendrocyte-specific mitochondrial DNA double-strand breaks in PLP:mtPstI mice cause oligodendrocyte death and demyelination associated with axonal damage and glial activation. Hence, PLP:mtPstI mice represent a unique tool to study the pathological consequences of mitochondrial dysfunction in oligodendrocytes, as well as an ideal platform to test remyelinating and neuroprotective agents.

Nuclear factor-kappa B (NF-κB) is a key modulator of inflammation and secondary injury responses in neurodegenerative disease, including spinal cord injury (SCI). Inhibition of astroglial NF-κB reduces inflammation, enhances oligodendrogenesis and improves functional recovery after SCI, however the contribution of neuronal NF-κB to secondary inflammatory responses following SCI has yet to be investigated. We demonstrate that conditional ablation of IKK2 in Synapsin 1-expressing neurons in mice (Syn1creIKK2fl/fl) reduces activation of the classical NF-κB signaling pathway, resulting in impaired motor function and altered memory retention under naïve conditions. Following induction of a moderate SCI phosphorylated NF-κB levels decreased in the spinal cord of Syn1creIKK2fl/fl mice compared to controls, resulting in improvement in functional recovery. Histologically, Syn1creIKK2fl/fl mice exhibited reduced lesion volume but comparable microglial/leukocyte responses after SCI. In parallel, interleukin (IL)-1β expression was significantly decreased within the lesioned spinal cord, whereas IL-5, IL-6, IL-10, tumor necrosis factor (TNF) and chemokine (C-X-C motif) ligand 1 were unchanged compared to control mice. We conclude that conditional ablation of IKK2 in neurons, resulting in reduced neuronal NF-B signaling, and lead to protective effects after SCI and propose the neuronal classical NF-κB pathway as a potential target for the development of new therapeutic, neuroprotective strategies for SCI.

Patients suffering from neuropathic pain have a higher incidence of mood disorders such as depression. Increased expression of tumor necrosis factor (TNF) has been reported in neuropathic pain and depressive-like conditions and most of the pro-inflammatory effects of TNF are mediated by the TNF receptor 1 (TNFR1). Here we sought to investigate: (1) the occurrence of depressive-like behavior in chronic neuropathic pain and the associated forms of hippocampal plasticity, and (2) the involvement of TNFR1-mediated TNF signaling as a possible regulator of such events. Neuropathic pain was induced by chronic constriction injury of the sciatic nerve in wild-type and TNFR1(-/-) mice. Anhedonia, weight loss and physical state were measured as symptoms of depression. Hippocampal neurogenesis, neuroplasticity, myelin remodeling and TNF/TNFRs expression were analyzed by immunohistochemical analysis and western blot assay. We found that neuropathic pain resulted in the development of depressive symptoms in a time dependent manner and was associated with profound hippocampal alterations such as impaired neurogenesis, reduced expression of neuroplasticity markers and myelin proteins. The onset of depressive-like behavior also coincided with increased hippocampal levels of TNF, and decreased expression of TNF receptor 2 (TNFR2), which were all fully restored after mice spontaneously recovered from pain. Notably, TNFR1(-/-) mice did not develop depressive-like symptoms after injury, nor were there changes in hippocampal neurogenesis and plasticity. Our data show that neuropathic pain induces a cluster of depressive-like symptoms and profound hippocampal plasticity that are dependent on TNF signaling through TNFR1.

Remyelination failure is a crucial component of disease progression in the autoimmune demyelinating disease Multiple Sclerosis (MS). The regenerative capacity of oligodendrocyte progenitor cells (OPCs) to replace myelinating oligodendrocytes is likely influenced by many aspects of the lesion environment including inflammatory signaling and extracellular matrix (ECM) deposition. These features of MS lesions are typically attributed to infiltrating leukocytes and reactive astrocytes. Here we demonstrate that fibroblasts also contribute to the inhibitory environment in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Using Col1α1GFPtransgenic mice, we show that perivascular fibroblasts are activated in the spinal cord at EAE onset, and infiltrate the parenchyma by the peak of behavioral deficits where they are closely associated with areas of demyelination, myeloid cell accumulation, and ECM deposition. We further show that both fibroblast conditioned media and fibroblast ECM inhibit the differentiation of OPCs into mature oligodendrocytes. Taken together, our results indicate that the fibrotic scar is a major component of EAE pathology that leads to an inhibitory environment for remyelination, thus raising the possibility that anti-fibrotic mechanisms may serve as novel therapeutic targets for MS.

Multiple sclerosis (MS), a neuroinflammatory disease, has few treatment options, none entirely adequate. We studied whether prolonged electrical microstimulation of a hindbrain region (the nucleus raphe magnus) can attenuate experimental autoimmune encephalomyelitis, a murine model of MS induced by MOG35-55 injection. Eight days after symptoms emerged, a wireless electrical stimulator with an attached microelectrode was implanted cranially, and daily intermittent stimulation was begun in awake, unrestrained mice. The thoracic spinal cord was analyzed for changes in histology (on day 29) and gene expression (on day 37), with a focus on myelination and cytokine production. Controls, with inactive implants, showed a phase of disease exacerbation on days 19–25 that stimulation for >16 days eliminated. Prolonged stimulation also reduced numbers of infiltrating immune cells and increased numbers of myelinated axons. It additionally lowered genetic expression of some pro-inflammatory cytokines (interferon gamma and tumor necrosis factor) and platelet-derived growth factor receptor alpha, a marker of oligodendrocyte precursors, while raising expression of myelin basic protein. Studies of restorative treatments for MS might profitably consider ways to stimulate the raphe magnus, directly or via its inputs, or to emulate its serotonergic and peptidergic output.

Microglia are activated following cerebral ischemia and increase their production of the neuro- and immunomodulatory cytokine tumor necrosis factor (TNF). To address the function of TNF from this cellular source in focal cerebral ischemia we used TNF conditional knock out mice (LysMcreTNFfl/fl) in which the TNF gene was deleted in cells of the myeloid lineage, including microglia. The deletion reduced secreted TNF levels in lipopolysaccharide-stimulated cultured primary microglia by ~93%. Furthermore, phosphorylated-ERK/ERK ratios were significantly decreased in naïve LysMcreTNFfl/fl mice demonstrating altered ERK signal transduction. Micro-PET using 18[F]-fluorodeoxyglucose immediately after focal cerebral ischemia showed increased glucose uptake in LysMcreTNFfl/fl mice, representing significant metabolic changes, that translated into increased infarct volumes at 24 hours and 5 days compared to littermates (TNFfl/fl). In naïve LysMcreTNFfl/fl mice cytokine levels were low and comparable to littermates. At 6 hours, TNF producing microglia were reduced by 56% in the ischemic cortex in LysMcreTNFfl/fl mice compared to littermate mice, whereas no TNF+ leukocytes were detected. At 24 hours, pro-inflammatory cytokine (TNF, IL-1β, IL-6, IL-5 and CXCL1) levels were significantly lower in LysMcreTNFfl/fl mice, despite comparable infiltrating leukocyte populations. Our results identify microglial TNF as beneficial and neuroprotective in the acute phase and as a modulator of neuroinflammation at later time points after experimental ischemia, which may contribute to regenerative recovery.

Background Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by motor and non-motor symptoms. The cause of the motor symptoms is the loss of dopaminergic neurons in the substantia nigra with consequent depletion of dopamine in the striatum. Although the etiology of PD is unknown, mitochondrial dysfunctions, including cytochrome c oxidase (Complex IV) impairment in dopaminergic neurons, have been associated with the disease’s pathophysiology. In order to analyze the role of Complex IV in PD, we knocked out Cox10 (essential for the maturation of COXI, a catalytic subunit of Complex IV) in dopaminergic neurons. We also tested whether the resulting phenotype was improved by stimulating the PPAR-γ pathway. Results Cox10/DAT-cre mice showed decreased numbers of TH+ and DAT+ cells in the substantia nigra, early striatal dopamine depletion, motor defects reversible with L-DOPA treatment and hypersensitivity to L-DOPA with hyperkinetic behavior. We found that chronic pioglitazone (PPAR-γ agonist) treatment ameliorated the motor phenotype in Cox10/DAT-cre mice. Although neither mitochondrial function nor the number of dopaminergic neurons was improved, neuroinflammation in the midbrain and the striatum was decreased. Conclusions By triggering a mitochondrial Complex IV defect in dopaminergic neurons, we created a new mouse model resembling the late stages of PD with massive degeneration of dopaminergic neurons and striatal dopamine depletion. The motor phenotypes were improved by Pioglitazone treatment, suggesting that targetable secondary pathways can influence the development of certain forms of PD. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0090-7) contains supplementary material, which is available to authorized users.

Traumatic spinal cord injury (SCI) is followed by an instant increase in expression of the microglial-derived proinflammatory cytokine tumor necrosis factor (TNF) within the lesioned cord. TNF exists both as membrane-anchored TNF (mTNF) and as cleaved soluble TNF (solTNF). We previously demonstrated that epidural administration of a dominant-negative inhibitor of solTNF, XPro1595, to the contused spinal cord resulted in changes in Iba1 protein expression in microglia/macrophages, decreased lesion volume, and improved locomotor function. Here, we extend our studies using mice expressing mTNF, but no solTNF (mTNFΔ/Δ), to study the effect of genetic ablation of solTNF on SCI. We demonstrate that TNF levels were significantly decreased within the lesioned spinal cord 3 days after SCI inmTNFΔ/Δmice compared to littermates. This decrease did, however, not translate into significant changes in other pro- and anti-inflammatory cytokines (IL-10, IL-1β, IL-6, IL-5, IL-2, CXCL1, CCL2, or CCL5), despite a tendency towards increased IL-10 and decreased IL-1β, TNFR1, and TNFR2 levels inmTNFΔ/Δmice. In addition, microglial and leukocyte infiltration, activation state (Iba1, CD11b, CD11c, CD45, and MHCII), lesion size, and functional outcome after moderate SCI were comparable between genotypes. Collectively, our data demonstrate that genetic ablation of solTNF does not significantly modulate postlesion outcome after SCI.

We utilized a transgenic mouse model where nuclear factor kappa B (NF-κB) is selectively inhibited in glial fibrillary acidic protein (GFAP) expressing cells. The transgene, GFAP-IκBα-dn, overexpresses a dominant negative form of the inhibitor of NF-κB (IκBα) under the control of the GFAP promoter. In the present work, we sought to understand the impact of glial NF-κB inhibition on the expression of pain mediating sensory neuropeptides galanin and calcitonin gene related peptide (CGRP) in a model of neuropathic pain in mice. Chronic constriction injury (CCI) of the left sciatic nerve was performed on wild type (WT) and GFAP-IκBα-dn transgenic mice. RT-PCR and immunohistological staining were performed in sciatic nerve and/or L4-L5 DRG tissue for galanin, CGRP and macrophage marker CD11b. GFAP-IκBα-dn mice had less mechanical and thermal hyperalgesia compared to WT mice post-CCI. After CCI, we observed galanin upregulation in DRG and sciatic nerve, which was less in GFAP-IκBα-dn mice. CGRP gene expression in the DRG increased transiently on day 1 post-CCI in WT but not in GFAP-IκBα-dn mice, and no evidence of CGRP upregulation in sciatic nerve post-CCI was found. After CCI, upregulation of CD11b in sciatic nerve was less in GFAP-IκBα-dn mice compared to WT mice, indicative of less macrophage infiltration. Our results showed that glial NF-κB inhibition reduces galanin and CGRP expression, which are neuropeptides that correlate with pain behavior and inflammation after peripheral nerve injury.

Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF-kappaB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia-reperfusion in wild-type (WT) and in GFAP-IkappaBalpha-dn transgenic mice, where the NF-kappaB classical pathway is suppressed specifically in astrocytes. The GFAP-IkappaBalpha-dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro-inflammatory genes, encoding Tnf-alpha, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO-synthase in the retinas of GFAP-IkappaBalpha-dn mice. These data suggest that certain NF-kappaB-regulated pro-inflammatory and redox-active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.

We previously showed that Nuclear Factor kappaB (NF-kappaB) inactivation in astrocytes leads to improved functional recovery following spinal cord injury (SCI). This correlated with reduced expression of pro-inflammatory mediators and chondroitin sulfate proteoglycans, and increased white matter preservation. Hence we hypothesized that inactivation of astrocytic NF-kappaB would create a more permissive environment for axonal sprouting and regeneration. We induced both contusive and complete transection SCI in GFAP-Inhibitor of kappaB-dominant negative (GFAP-IkappaBalpha-dn) and wild-type (WT) mice and performed retrograde [fluorogold (FG)] and anterograde [biotinylated dextran amine (BDA)] tracing 8 weeks after injury. Following contusive SCI, more FG-labeled cells were found in motor cortex, reticular formation, and raphe nuclei of transgenic mice. Spared and sprouting BDA-positive corticospinal axons were found caudal to the lesion in GFAP-IkappaBalpha-dn mice. Higher numbers of FG-labeled neurons were detected immediately rostral to the lesion in GFAP-IkappaBalpha-dn mice, accompanied by increased expression of synaptic and axonal growth-associated molecules. After transection, however, no FG-labeled neurons or BDA-filled axons were found rostral and caudal to the lesion, respectively, in either genotype. These data demonstrated that inhibiting astroglial NF-kappaB resulted in a growth-supporting terrain promoting sparing and sprouting, rather than regeneration, of supraspinal and propriospinal circuitries essential for locomotion, hence contributing to the improved functional recovery observed after SCI in GFAP-IkappaBalpha-dn mice.

In the CNS, the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects. In this study, we show that transgenic inactivation of astroglial NF-κB (glial fibrillary acidic protein-IκBα-dominant-negative mice) resulted in reduced disease severity and improved functional recovery following experimental autoimmune encephalomyelitis. At the chronic stage of the disease, transgenic mice exhibited an overall higher presence of leukocytes in spinal cord and brain, and a markedly higher percentage of CD8+CD122+ T regulatory cells compared with wild type, which correlated with the timing of clinical recovery. We also observed that expression of proinflammatory genes in both spinal cord and cerebellum was delayed and reduced, whereas the loss of neuronal-specific molecules essential for synaptic transmission was limited compared with wild-type mice. Furthermore, death of retinal ganglion cells in affected retinas was almost abolished, suggesting the activation of neuroprotective mechanisms. Our data indicate that inhibiting NF-κB in astrocytes results in neuroprotective effects following experimental autoimmune encephalomyelitis, directly implicating astrocytes in the pathophysiology of this disease.

Microglia respond to focal cerebral ischemia by increasing their production of the neuromodulatory cytokine tumor necrosis factor, which exists both as membrane-anchored tumor necrosis factor and as cleaved soluble tumor necrosis factor forms. We previously demonstrated that tumor necrosis factor knockout mice display increased lesion volume after focal cerebral ischemia, suggesting that tumor necrosis factor is neuroprotective in experimental stroke. Here, we extend our studies to show that mice with intact membrane-anchored tumor necrosis factor, but no soluble tumor necrosis factor, display reduced infarct volumes at one and five days after stroke. This was associated with improved functional outcome after experimental stroke. No changes were found in the mRNA levels of tumor necrosis factor and tumor necrosis factor-related genes (TNFR1, TNFR2, TACE), pro-inflammatory cytokines (IL-1β, IL-6) or chemokines (CXCL1, CXCL10, CCL2); however, protein expression of TNF, IL-1β, IL-6 and CXCL1 was reduced in membrane-anchored tumor necrosis factorΔ/Δ compared to membrane-anchored tumor necrosis factorwt/wt mice one day after experimental stroke. This was paralleled by reduced MHCII expression and a reduction in macrophage infiltration in the ipsilateral cortex of membrane-anchored tumor necrosis factorΔ/Δ mice. Collectively, these findings indicate that membrane-anchored tumor necrosis factor mediates the protective effects of tumor necrosis factor signaling in experimental stroke, and therapeutic strategies specifically targeting soluble tumor necrosis factor could be beneficial in clinical stroke therapy.

Contains fulltext : 50264tits.pdf (Publisher’s version ) (Closed access) OBJECTIVES: To develop and validate a novel genotyping approach, named infrared Fluorescence Allele Specific Hybridization (iFLASH), which combines the principles of allele specific oligonucleotide (ASO) hybridization with the advanced possibilities of infrared imaging. DESIGN AND METHODS: As an example, we genotyped the 55L > M and the 192Q > R common genetic variants of the paraoxonase-1 gene in 92 DNA samples using the iFLASH technique, and validated the outcomes with the restriction fragment length polymorphism (RFLP) and TAQman genotyping assays. RESULTS: There was a 100 percent agreement in genotype outcome among the three methods. CONCLUSIONS: Although we found complete unity in genotype outcome, the iFLASH assay has essential advantages over the RFLP and TAQman genotyping assays. First, the iFLASH technique is capable of handling up to 1536 samples per assay, which makes it a suitable technique for high-throughput genotyping. Secondly, because the costs per assay are lower, high-throughput genotyping with iFLASH is affordable.

The transcription factor NF-κB plays an important role in both physiological and pathological events in the central nervous system. Nevertheless, the mechanisms of NF-κB-mediated regulation of gene expression, and the signaling molecules participating in the NF-κB pathway in the central nervous system are, to date, poorly understood. To identify such molecules, we conducted a yeast two-hybrid screen of a human brain cDNA library using NIK as bait. As a result, we identified a novel NIK and IKKβ binding protein designated NIBP that is mainly expressed in brain, muscle, heart, and kidney. Interestingly, low levels of expression were detected in immune tissues such as spleen, thymus, and peripheral blood leukocytes, where NF-κB is known to modulate immune function. We demonstrated by immunohistochemistry that NIBP expression in the brain is localized to neurons. NIBP physically interacts with NIK, IKKβ, but not IKKα or IKKγ. NIBP overexpression potentiates tumor necrosis factor-α-induced NF-κB activation through increased phosphorylation of the IKK complex and its downstream IκBα and p65 substrates. Finally, knockdown of NIBP expression by small interfering RNA reduces tumor necrosis factor-α-induced NF-κB activation, prevents nerve growth factor-induced neuronal differentiation, and decreases Bcl-xL gene expression in PC12 cells. Our data demonstrate that NIBP, by interacting with NIK and IKKβ, is a new enhancer of the cytokine-induced NF-κB signaling pathway. Because of its neuronal expression, we propose that NIBP may be a potential target for modulating the NF-κB signaling cascade in neuronal pathologies dependent upon abnormal activation of this pathway.