Tlr4 Deletion Modulates Cytokine and Extracellular Matrix Expression in Chronic Spinal Cord Injury, Leading to Improved Secondary Damage and Functional Recovery.

Toll-like receptors (TLRs) play an important role in the innate immune response after CNS injury. Although TLR4 is one of the best characterized, its role in chronic stages after spinal cord injury (SCI) is not well understood. We examined the role of TLR4 signaling in injury-induced responses at 1 d, 7 d, and 8 weeks after spinal cord contusion injury in adult female TLR4 null and wild-type mice. Analyses include secondary damage, a range of transcriptome and protein analyses of inflammatory, cell death, and extracellular matrix (ECM) molecules, as well as immune cell infiltration and changes in axonal sprouting and locomotor recovery. Lack of TLR4 signaling results in reduced neuronal and myelin loss, reduced activation of NFκB, and decreased expression of inflammatory cytokines and necroptotic cell death pathway at a late time point (8 weeks) after injury. TLR4 null mice also showed reduction of scar-related ECM molecules at 8 weeks after SCI, accompanied by increase in ECM molecules associated with perineuronal nets, increased sprouting of serotonergic fibers, and improved locomotor recovery. These findings reveal novel effects of TLR4 signaling in chronic SCI. We show that TLR4 influences inflammation, cell death, and ECM deposition at late-stage post-injury when secondary injury processes are normally considered to be over. This highlights the potential for late-stage targeting of TLR4 as a potential therapy for chronic SCI.

Ferroptosis induces detrimental effects in chronic EAE and its implications for progressive MS.

Ferroptosis is a form of lipid peroxidation-mediated cell death and damage triggered by excess iron and insufficiency in the glutathione antioxidant pathway. Oxidative stress is thought to play a crucial role in progressive forms of multiple sclerosis (MS) in which iron deposition occurs. In this study we assessed if ferroptosis plays a role in a chronic form of experimental autoimmune encephalomyelitis (CH-EAE), a mouse model used to study MS. Changes were detected in the mRNA levels of several ferroptosis genes in CH-EAE but not in relapsing-remitting EAE. At the protein level, expression of iron importers is increased in the earlier stages of CH-EAE (onset and peak). While expression of hemoxygenase-1, which mobilizes iron from heme, likely from phagocytosed material, is increased in macrophages at the peak and progressive stages. Excess iron in cells is stored safely in ferritin, which increases with disease progression. Harmful, redox active iron is released from ferritin when shuttled to autophagosomes by 'nuclear receptor coactivator 4' (NCOA4). NCOA4 expression increases at the peak and progressive stages of CH-EAE and accompanied by increase in redox active ferrous iron. These changes occur in parallel with reduction in the antioxidant pathway (system xCT, glutathione peroxidase 4 and glutathione), and accompanied by increased lipid peroxidation. Mice treated with a ferroptosis inhibitor for 2 weeks starting at the peak of CH-EAE paralysis, show significant improvements in function and pathology. Autopsy samples of tissue sections of secondary progressive MS (SPMS) showed NCOA4 expression in macrophages and oligodendrocytes along the rim of mixed active/inactive lesions, where ferritin+ and iron containing cells are located. Cells expressing NCOA4 express less ferritin, suggesting ferritin degradation and release of redox active iron, as indicated by increased lipid peroxidation. These data suggest that ferroptosis is likely to contribute to pathogenesis in CH-EAE and SPMS.

Drug screening in zebrafish larvae reveals inflammation-related modulators of secondary damage after spinal cord injury in mice.

Prolonged inflammation after spinal cord injury is detrimental to recovery. To find pharmacological modulators of the inflammation response, we designed a rapid drug screening paradigm in larval zebrafish followed by testing of hit compounds in a mouse spinal cord injury model. Methods: We used reduced il-1ß linked green fluorescent protein (GFP) reporter gene expression as a read-out for reduced inflammation in a screen of 1081 compounds in larval zebrafish. Hit drugs were tested in a moderate contusion model in mice for cytokine regulation, and improved tissue preservation and locomotor recovery. Results: Three compounds robustly reduced il-1ß expression in zebrafish. Cimetidine, an over-the-counter H2 receptor antagonist, also reduced the number of pro-inflammatory neutrophils and rescued recovery after injury in a zebrafish mutant with prolonged inflammation. Cimetidine action on il-1ß expression levels was abolished by somatic mutation of H2 receptor hrh2b, suggesting specific action. In mice, systemic treatment with Cimetidine led to significantly improved recovery of locomotor behavior as compared to controls, accompanied by decreased neuronal tissue loss and a shift towards a pro-regenerative profile of cytokine gene expression. Conclusion: Our screen revealed H2 receptor signaling as a promising target for future therapeutic interventions in spinal cord injury. This work highlights the usefulness of the zebrafish model for rapid screening of drug libraries to identify therapeutics to treat mammalian spinal cord injury.

Ferroptosis in Neurological Disease.

Iron accumulation in the CNS occurs in many neurological disorders. It can contribute to neuropathology as iron is a redox-active metal that can generate free radicals. The reasons for the iron buildup in these conditions are varied and depend on which aspects of iron influx, efflux, or sequestration that help maintain iron homeostasis are dysregulated. Iron was shown recently to induce cell death and damage via lipid peroxidation under conditions in which there is deficient glutathione-dependent antioxidant defense. This form of cell death is called ferroptosis. Iron chelation has had limited success in the treatment of neurological disease. There is therefore much interest in ferroptosis as it potentially offers new drugs that could be more effective in reducing iron-mediated lipid peroxidation within the lipid-rich environment of the CNS. In this review, we focus on the molecular mechanisms that induce ferroptosis. We also address how iron enters and leaves the CNS, as well as the evidence for ferroptosis in several neurological disorders. Finally, we highlight biomarkers of ferroptosis and potential therapeutic strategies.

The impact of brain lesions on sexual dysfunction in patients with multiple sclerosis: A systematic review of magnetic resonance imaging studies.

BACKGROUND: Sexual dysfunction is common but underestimated clinical symptom in MS patients. A growing body of evidence has been suggested the link between brain lesions and sexual dysfunction (SD) in patients with multiple sclerosis (MS). However, the clinical research investigating this relationship have shown inconsistent results. Here, we aimed to systematically review the magnetic resonance imaging (MRI) studies evaluating the association between the brain lesions and SD in MS patients. METHODS: This study was provided according to the recommendations of the preferred reporting items for systematic reviews and meta-analyses statement. A comprehensive systematic search of online databases was performed to find eligible studies up to December 2020. The quality of studies was methodologically assessed using Newcastle-Ottawa Scale score. RESULTS: We identified eight articles regarding MS brain lesions and SD through the search strategy. Seven studies showed significant associations between SD and brain lesions. Three studies investigated the brain stem, two studies the insular and occipital region, one study the frontal lobe, prefrontal cortex, and temporal lobe and one study the parietal area. CONCLUSION: The results of this systematic review showed that lesions in different brain areas are correlated with SD in MS patients. Plaques in the occipital and hippocampus areas, as well as left insula appear to be related to dysfunction of sexual arousability or lubrication/erection in MS patients. Orgasmic dysfunction in MS patients may be associated with brain lesions in pons, left temporal periventricular, and right occipital areas.

MicroRNA-210 regulates the metabolic and inflammatory status of primary human astrocytes.

BACKGROUND: Astrocytes are the most numerous glial cell type with important roles in maintaining homeostasis and responding to diseases in the brain. Astrocyte function is subject to modulation by microRNAs (miRs), which are short nucleotide strands that regulate protein expression in a post-transcriptional manner. Understanding the miR expression profile of astrocytes in disease settings provides insight into the cellular stresses present in the microenvironment and may uncover pathways of therapeutic interest. METHODS: Laser-capture microdissection was used to isolate human astrocytes surrounding stroke lesions and those from neurological control tissue. Astrocytic miR expression profiles were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Primary human fetal astrocytes were cultured under in vitro stress conditions and transfection of a miR mimic was used to better understand how altered levels of miR-210 affect astrocyte function. The astrocytic response to stress was studied using qPCR, enzyme-linked immunosorbent assays (ELISAs), measurement of released lactate, and Seahorse. RESULTS: Here, we measured miR expression levels in astrocytes around human ischemic stroke lesions and observed differential expression of miR-210 in chronic stroke astrocytes compared to astrocytes from neurological control tissue. We also identified increased expression of miR-210 in mouse white matter tissue around middle cerebral artery occlusion (MCAO) brain lesions. We aimed to understand the role of miR-210 in primary human fetal astrocytes by developing an in vitro assay of hypoxic, metabolic, and inflammatory stresses. A combination of hypoxic and inflammatory stresses was observed to upregulate miR-210 expression. Transfection with miR-210-mimic (210M) increased glycolysis, enhanced lactate export, and promoted an anti-inflammatory transcriptional and translational signature in astrocytes. Additionally, 210M transfection resulted in decreased expression of complement 3 (C3) and semaphorin 5b (Sema5b). CONCLUSIONS: We conclude that miR-210 expression in human astrocytes is modulated in response to ischemic stroke disease and under in vitro stress conditions, supporting a role for miR-210 in the astrocytic response to disease conditions. Further, the anti-inflammatory and pro-glycolytic impact of miR-210 on astrocytes makes it a potential candidate for further research as a neuroprotective agent.

Dysregulation of Iron Homeostasis in the Central Nervous System and the Role of Ferroptosis in Neurodegenerative Disorders.

Significance: Iron accumulation occurs in the central nervous system (CNS) in a variety of neurological conditions as diverse as spinal cord injury, stroke, multiple sclerosis, Parkinson's disease, and others. Iron is a redox-active metal that gives rise to damaging free radicals if its intracellular levels are not controlled or if it is not properly sequestered within cells. The accumulation of iron occurs due to dysregulation of mechanisms that control cellular iron homeostasis. Recent Advances: The molecular mechanisms that regulate cellular iron homeostasis have been revealed in much detail in the past three decades, and new advances continue to be made. Understanding which aspects of iron homeostasis are dysregulated in different conditions will provide insights into the causes of iron accumulation and iron-mediated tissue damage. Recent advances in iron-dependent lipid peroxidation leading to cell death, called ferroptosis, has provided useful insights that are highly relevant for the lipid-rich environment of the CNS. Critical Issues: This review examines the mechanisms that control normal cellular iron homeostasis, the dysregulation of these mechanisms in neurological disorders, and more recent work on how iron can induce tissue damage via ferroptosis. Future Directions: Quick and reliable tests are needed to determine if and when ferroptosis contributes to the pathogenesis of neurological disorders. In addition, there is need to develop better druggable agents to scavenge lipid radicals and reduce CNS damage for neurological conditions for which there are currently few effective treatments. Antioxid. Redox Signal. 37, 150-170.

How cytosolic compartments play safeguard functions against neuroinflammation and cell death in cerebral ischemia.

Ischemic stroke is the second leading cause of mortality and disability globally. Neuronal damage following ischemic stroke is rapid and irreversible, and eventually results in neuronal death. In addition to activation of cell death signaling, neuroinflammation is also considered as another pathogenesis that can occur within hours after cerebral ischemia. Under physiological conditions, subcellular organelles play a substantial role in neuronal functionality and viability. However, their functions can be remarkably perturbed under neurological disorders, particularly cerebral ischemia. Therefore, their biochemical and structural response has a determining role in the sequel of neuronal cells and the progression of disease. However, their effects on cell death and neuroinflammation, as major underlying mechanisms of ischemic stroke, are still not understood. This review aims to provide a comprehensive overview of the contribution of each organelle on these pathological processes after ischemic stroke.

Neurochemicals of limbic system and thalamofrontal cortical network: Are they different between patients with idiopathic generalized epilepsy and psychogenic nonepileptic seizure?

OBJECTIVE: Thalamofrontal cortical network and limbic system are proposed to be involved in psychogenic nonepileptic seizure (PNES) and idiopathic generalized epilepsy (IGE). This study aimed to investigate neurochemical changes in prefrontal cortex, thalamus, and limbic circuits in patients with PNES and IGE. We also analyzed the interaction between cognitive functions and neurochemical changes in both groups. METHODS: Hydrogen proton magnetic resonance spectroscopy (1H-MRS) was used to measure N-acetyl aspartate (NAA), choline (Cho), creatine (Cr), glutamate-glutamine (Glx), and myo-inositol (MI). The voxels were placed on the bilateral dorsolateral prefrontal cortex (DLPFC), dorsomedial prefrontal cortex (DMPFC), anterior cingulate cortex (ACC), and thalamus. Attention and inhibitory control, as well as general intelligence status, were investigated using the Integrated Visual and Auditory Continuous Performance Test (IVA-CPT) and the Wechsler Adult Intelligence Scale (WAIS), respectively, in patients with PNES and IGE, as well as healthy volunteers. RESULTS: The 1H-MRS showed a decreased ratio of NAA/Cr in the right and left thalamus, right DMPFC, and right ACC in patients with IGE and PNES. Furthermore, a decrease of the NAA/Cr ratio in the left DMPFC and an increase of NAA/Cr ratio in the right DLPFC were observed in patients with PNES compared with the controls. The patient groups had a decreased ratio of Cho/Cr in right ACC compared with the healthy subjects. Moreover, the NAA/Cr ratio in the left thalamus and left DMPFC was correlated with seizure frequency in patient groups. Reduced NAA/Cr ratio in the right ACC and left DLPFC were also correlated with poor IVA-CPT scores. CONCLUSION: This study highlighted the dysfunction in prefrontal-thalamic-limbic circuits and impairment in neurocognition in patients with PNES and IGE.

Elevated Blood-Based Brain Biomarker Levels in Patients with Epileptic Seizures: A Systematic Review and Meta-analysis.

Recently, growing attention has been paid to the changes of brain biomarkers following the epilepsy. However, establishing specific epilepsy-related biomarkers has been impeded due to contradictory findings. This study systematically reviewed the evidence on brain biomarkers in epilepsy and determined reliable biomarkers in epileptic patients. A comprehensive systematic search of online databases was performed to find eligible studies up to August 2019. The quality of studies methodologically was assessed using the Newcastle-Ottawa Scale score. Among the several biomarkers, S100 calcium binding protein B (S100B) and neuron specific enolase (NSE) have been qualified for meta-analysis of the association between epilepsy and the brain biomarkers. Inverse-variance weights method was used to calculate pooled standardized mean difference (SMD) estimate with 95% CI, and random effects meta-analysis was conducted taking into account conceptual heterogeneity. Sensitivity analysis and publication bias assessment was performed using Stata. Of 29 studies that were qualified for further analysis, only 22 studies were eligible to quantify by meta-analysis. Significant increase of serum S100B levels (SMD = 0.80; 95% CI 0.18 to 1.42) but not NSE (SMD = 0.45; 95% CI -0.09 to 1.00) has been found in epileptic patients compared with healthy controls. Subgroup meta-analysis by age demonstrated that S100B could be found in pediatric (SMD = 1.15; 95% CI 0.03 to 2.27) not adult patients (SMD = 0.43; 95% CI -0.12 to 0.98). Findings of this meta-analysis indicate that serum level of S100B is significantly increased in epileptic patients, suggesting the elevation and release of the brain biomarkers from brain to blood following epileptic seizures.

Coenzyme Q10 Insufficiency Contributes to the Duration and Frequency of Seizures in Epileptic Patients.

INTRODUCTION: Oxidative stress has recently emerged as a possible mechanism in the pathogenesis of epilepsy. Coenzyme Q10 (CoQ10) is a strong endogenous antioxidant that protects cells from lipid oxidation and Reactive Oxygen Species (ROS) production; however, the impact of CoQ10 on seizure characteristics in epileptic patients is unclear. METHODS: The current study enrolled patients with Epileptic Seizure (ES) to evaluate their serum concentration of CoQ10 and to investigate whether a relationship exists between CoQ10 levels with the duration, frequency, and type of seizure. RESULTS: A total of 39 patients with epileptic seizures and 35 healthy controls were included in the study. The levels of CoQ10 in ES patients were significantly lower in comparison with healthy controls (11.99±5.93 vs (ng/ml). 16.48±4.20 (ng/ml) P<0.001). We also found that the duration of epilepsy and seizure frequency was negatively correlated with serum CoQ10 levels. CONCLUSION: These findings indicate that CoQ10 deficiency might substantially contribute to the clinical signs of epileptic patients.

Serum Coenzyme Q10 Is Associated with Clinical Neurological Outcomes in Acute Stroke Patients.

Disruption of prooxidant-antioxidant balance may lead to oxidative stress which is known as a mechanism contributing to ischemic stroke. Coenzyme Q10 (CoQ10) is an endogenous antioxidant that could be effective in preventing oxidative stress. However, the contribution of serum levels of CoQ10 in clinical neurological outcomes following ischemic stroke has not been clearly established. This study aims at measuring serum concentration of CoQ10 along with major indicators of antioxidant and oxidant among patients within 24 h after onset of the stroke symptoms, and investigating their relation with the clinical status of patients. Serum levels of CoQ10, superoxide dismutase (SOD), and malondialdehyde (MDA) were measured in 76 patients and 34 healthy individuals. Severity of the neurological deficit, functional disability, and cognitive status in ischemic subjects were respectively studied with the National Institutes of Health stroke scale (NIHSS), modified Rankin Scale (MRS), and Mini-Mental State Examination (MMSE). Stroke patients had significantly lower serum level of CoQ10 and SOD as compared to controls (27.34 ± 35.40 ng/ml, 18.58 ± 0.76 µ/ml, respectively; p < 0.05), whereas the serum MDA level was significantly higher (38.02 ± 2.61 µm, p < 0.05). A significant negative correlation was detected between the serum CoQ10 level and scores of NIHSS and MRS. A similar association was discerned between the SOD level and the neurological deficit score. The serum MDA level was also found to be strongly correlated with all three neurological scales. These findings suggest that the serum level of CoQ10 like other antioxidant and oxidant markers can significantly change early after ischemic stroke and they are substantially associated with clinical neurological outcomes.

Temporal Pattern and Crosstalk of Necroptosis Markers with Autophagy and Apoptosis Associated Proteins in Ischemic Hippocampus.

Necroptosis, a novel type of programmed cell death, has been recently implicated as a possible mechanism for cerebral ischemia-reperfusion (I/R) injury. We herein studied time-dependent changes of necroptosis markers along with apoptosis- and autophagy-associated proteins in rat hippocampus at 1, 3, 6, 12, 24, and 48 h after global cerebral I/R injury. Furthermore, to determine the cross talk between autophagy and necroptosis, we examined the effects of pretreatment with bafilomycin-A1 (Baf-A1), as a late-stage autophagy inhibitor, on necroptosis. Highest levels of receptor-interacting protein 1 and 3 (RIP1 and RIP3), as key mediators of necroptosis, were observed at 24 h after reperfusion. Alongside, activity of glutamate dehydrogenase (GLUD1), downstream enzyme of RIP3, was increased. Peak time of necroptosis was subsequent to caspase-3-dependent cell death that peaked at 12 h of reperfusion but concurrent with autophagy. Administration of Baf-A1 could attenuate necroptosis, verified by decrease in RIP1 and RIP3 protein levels, as well as GLUD1 activity. However, there was no significant change in caspase-3-dependent cell death. Taken together, our results highlight that global cerebral I/R activates necroptosis that could be triggered by autophagy and interacts reversely with caspase-3-dependent apoptosis.

Ceruloplasmin Plays a Neuroprotective Role in Cerebral Ischemia.

Ceruloplasmin (Cp) is a ferroxidase that also plays a role in iron efflux from cells. It can thus help to regulate cellular iron homeostasis. In the CNS, Cp is expressed as a membrane-anchored form by astrocytes. Here, we assessed the role of Cp in permanent middle cerebral artery occlusion (pMCAO) comparing wildtype and Cp null mice. Our studies show that the lesion size is larger and functional recovery impaired in Cp null mice compared to wildtype mice. Expression of Cp increased ninefold at 72 h after pMCAO and remained elevated about twofold at day 14. We also assessed changes in mRNA and protein expression of molecules involved in iron homeostasis. As expected there was a reduction in ferroportin in Cp null mice at 72 h. There was also a remarkable increase in DMT1 protein in both genotypes at 72 h, being much higher in wildtype mice (19.5-fold), that then remained elevated about twofold at 14 days. No difference was seen in transferrin receptor 1 (TfR1) expression, except a small reduction in wildtype mice at 72 h, suggesting that the increase in DMT1 may underlie iron uptake independent of TfR1-endosomal uptake. There was also an increase of ferritin light chain in both genotypes. Iron histochemistry showed increased iron accumulation after pMCAO, initially along the lesion border and later throughout the lesion. Immunofluorescence labeling for ferritin (a surrogate marker for iron) and GFAP or CD11b showed increased ferritin in GFAP+ astrocytes along the lesion border in Cp null mice, while CD11b+ macrophages expressed ferritin equally in both genotypes. Increased lipid peroxidation assessed by 4HNE staining was increased threefold in Cp null mice at 72 h after pMCAO; and 3-nitrotyrosine labeling showed a similar trend. Three key pro-inflammatory cytokines (IL-1ß, TNFα, and IL-6) were markedly increased at 24 h after pMCAO equally in both genotypes, and remained elevated at lower levels later, indicating that the lack of Cp does not alter key inflammatory cytokine expression after pMCAO. These data indicate that Cp expression is rapidly upregulated after pMCAO, and loss of Cp results in dysregulation of iron homeostasis, increased oxidative damage, greater lesion size and impaired recovery of function.