Search

Neurologic disorders such as traumatic brain injury, multiple sclerosis, Alzheimer's disease, and drug-resistant epilepsy have a high socioeconomic impact around the world. Current therapies for these disorders are often not effective. This creates a demand for the development of new therapeutic approaches to treat these disorders. Recent data suggest that autoreactive naturally occurring immunoglobulins produced by subsets of B cells, called B1 B cells, combined with complement, are actively involved in the processes of restoration of neuronal functions during pathological conditions and remyelination. The focus of this review is to discuss the possibility of creating specific therapeutic antibodies that can activate and fix complement to enhance neuronal survival and promote central nervous system repair after injuries associated with many types of neurodegenerative diseases.

Complement system plays an important role in the immune defense against pathogens; however, recent studies demonstrated an important role of complement subunits C1q, C4, and C3 in normal functions of the central nervous system (CNS) such as non-functional synapse elimination (synapse pruning), and during various neurologic pathologies. Humans have two forms of C4 protein encoded by C4A and C4B genes that share 99.5% homology, while mice have only one C4B gene that is functionally active in the complement cascade. Overexpression of the human C4A gene was shown to contribute to the development of schizophrenia by mediating extensive synapse pruning through the activation C1q-C4-C3 pathway, while C4B deficiency or low levels of C4B expression were shown to relate to the development of schizophrenia and autism spectrum disorders possibly via other mechanisms not related to synapse elimination. To investigate the potential role of C4B in neuronal functions not related to synapse pruning, we compared wildtype (WT) mice with C3- and C4B- deficient animals for their susceptibility to pentylenetetrazole (PTZ)- induced epileptic seizures. We found that C4B (but not C3)-deficient mice were highly susceptible to convulsant and subconvulsant doses of PTZ when compared to WT controls. Further gene expression analysis revealed that in contrast to WT or C3-deficient animals, C4B-deficient mice failed to upregulate expressions of multiple immediate early genes (IEGs) Egrs1-4, c-Fos, c-Jus, FosB, Npas4, and Nur77 during epileptic seizures. Moreover, C4B-deficient mice had low levels of baseline expression of Egr1 on mRNA and protein levels, which was correlated with the cognitive problems of these animals. C4-deficient animals also failed to upregulate several genes downstream of IEGs such as BDNF and pro-inflammatory cytokines IL-1β, IL-6, and TNF. Taken together, our study demonstrates a new role of C4B in the regulation of expression of IEGs and their downstream targets during CNS insults such as epileptic seizures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Veremeyko, Jiang, He and Ponomarev.)

Cyanobacteria produce a wide range of structurally diverse cyanotoxins and bioactive cyanopeptides in freshwater, marine, and terrestrial ecosystems. The health significance of these metabolites, which include genotoxic- and neurotoxic agents, is confirmed by continued associations between the occurrence of animal and human acute toxic events and, in the long term, by associations between cyanobacteria and neurodegenerative diseases. Major mechanisms related to the neurotoxicity of cyanobacteria compounds include (1) blocking of key proteins and channels; (2) inhibition of essential enzymes in mammalian cells such as protein phosphatases and phosphoprotein phosphatases as well as new molecular targets such as toll-like receptors 4 and 8. One of the widely discussed implicated mechanisms includes a misincorporation of cyanobacterial non-proteogenic amino acids. Recent research provides evidence that non-proteinogenic amino acid BMAA produced by cyanobacteria have multiple effects on translation process and bypasses the proof-reading ability of the aminoacyl-tRNA-synthetase. Aberrant proteins generated by non-canonical translation may be a factor in neuronal death and neurodegeneration. We hypothesize that the production of cyanopeptides and non-canonical amino acids is a more general mechanism, leading to mistranslation, affecting protein homeostasis, and targeting mitochondria in eukaryotic cells. It can be evolutionarily ancient and initially developed to control phytoplankton communities during algal blooms. Outcompeting gut symbiotic microorganisms may lead to dysbiosis, increased gut permeability, a shift in blood-brain-barrier functionality, and eventually, mitochondrial dysfunction in high-energy demanding neurons. A better understanding of the interaction between cyanopeptides metabolism and the nervous system will be crucial to target or to prevent neurodegenerative diseases.

The glymphatic-lymphatic fluid transport system (GLFTS) consists of glymphatic pathway and cerebrospinal fluid (CSF) lymphatic outflow routes, allowing biological liquids from the brain parenchyma to access the CSF along with perivascular space and to be cleaned out of the skull through lymphatic vessels. It is known that increased local pressure due to physical compression of tissue improves lymphatic transport in peripheral organs, but little is known about the exact relationship between increased intracranial pressure (IICP) and GLFTS. In this study, we verify our hypothesis that IICP significantly impacts GLFTS, and this effect depends on severity of the IICP. Using a previously developed inflating balloon model to induce IICP and inject fluorescent tracers into the cisterna magna, we found significant impairment of the glymphatic circulation after IICP. We further found that cerebrovascular occlusion occurred, and cerebrovascular pulsation decreased after IICP. IICP also interrupted the drainage of deep cervical lymph nodes and dorsal meningeal lymphatic function, enhancing spinal lymphatic outflow to the sacral lymph nodes. Notably, these effects were associated with the severity of IICP. Thus, our findings proved that the intensity of IICP significantly impacts GLFTS. This may have translational applications for preventing and treating related neurological disorders.

Introduction: Coagulation parameters are important determinants for COVID-19 infection. We conducted meta-analysis to assess the association between early hemostatic parameters and infection severity., Methods: Electronic search was made for papers that addressed clinical characteristics of COVID-19 patients and disease severity. Results were filtered using exclusion and inclusion criteria and then pooled into a meta-analysis to estimate the standardized mean difference (SMD) with 95% confidence interval (CI) for D-dimers, fibrinogen, prothrombin time, platelet count (PLT), activated partial thromboplastin time. To explore the heterogeneity and robustness of our fundings, sensitivity and subgroup analyses were conducted. Publication bias was assessed with contour-enhanced funnel plots and Egger's test by linear regression. Coagulation parameters data from retrospective cohort study of 451 patients with COVID-19 at National Research Center for Cardiac Surgery were included in meta-analysis of published studies., Results: Overall, 41 original studies (17,601 patients) on SARS-CoV-2 were included. For the two groups of patients, stratified by severity, we identified that D-dimers, fibrinogen, activated partial thromboplastin time, and prothrombin time were significantly higher in the severe group [SMD 0.6985 with 95%CI (0.5155; 0.8815); SMD 0.661 with 95%CI (0.3387; 0.9833); SMD 0.2683 with 95%CI (0.1357; 0.4009); SMD 0.284 with 95%CI (0.1472; 0.4208)]. In contrast, PLT was significantly lower in patients with more severe cases of COVID-19 [SMD -0.1684 with 95%CI (-0.2826; -0.0542)]. Neither the analysis by the leave-one-out method nor the influence diagnostic have identified studies that solely cause significant change in the effect size estimates. Subgroup analysis showed no significant difference between articles originated from different countries but revealed that severity assessment criteria might have influence over estimated effect sizes for platelets and D-dimers. Contour-enhanced funnel plots and the Egger's test for D-dimers and fibrinogen revealed significant asymmetry that might be a sign of publication bias., Conclusions: The hemostatic laboratory parameters, with exception of platelets, are significantly elevated in patients with severe COVID-19. The two variables with strongest association to disease severity were D-dimers and fibrinogen levels. Future research should aim outside conventional coagulation tests and include analysis of clotting formation and platelet/platelet progenitors characteristics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Len, Iskakova, Sautbayeva, Kussanova, Tauekelova, Sugralimova, Dautbaeva, Abdieva, Ponomarev, Tikhonov, Bekbossynova and Barteneva.)

A deficiency in GM3-derived gangliosides, resulting from a lack of lactosylceramide-alpha-2,3-sialyltransferase (ST3GAL5), leads to severe neuropathology, including epilepsy and metabolic abnormalities. Disruption of ganglioside production by this enzyme may also have a role in the development of neuropsychiatric disorders. ST3Gal5 knock-out ( St3gal5 -/- ) mice lack a-, b-, and c-series gangliosides, but exhibit no overt neuropathology, possibly owing to the production of compensatory 0-series glycosphingolipids. Here, we sought to investigate the possibility that St3gal5 -/- mice might exhibit attention-deficit/hyperactivity disorder (ADHD)-like behaviours. In addition, we evaluated potential metabolic and electroencephalogram (EEG) abnormalities. St3gal5 -/- mice were subjected to behavioural testing, glucose tolerance tests, and the levels of expression of brain and peripheral A and B isoforms of the insulin receptor (IR) were measured. We found that St3gal5 -/- mice exhibit locomotor hyperactivity, impulsivity, neophobia, and anxiety-like behavior. The genotype also altered blood glucose levels and glucose tolerance. A sex bias was consistently found in relation to body mass and peripheral IR expression. Analysis of the EEG revealed an increase in amplitude in St3gal5 -/- mice. Together, St3gal5 -/- mice exhibit ADHD-like behaviours, altered metabolic and EEG measures providing a useful platform for better understanding of the contribution of brain gangliosides to ADHD and associated comorbidities.

Gangliosides are glycosphingolipids, which are abundant in brain, are known to modulate ion channels and cell-to-cell communication. Deficiencies can result in aberrant myelination and altered immune responses, which can give rise to neurodevelopmental psychiatric disorders. However, to date, little mechanistic data is available on how ganglioside deficiencies contribute to the behavioural disorders. In humans, the loss of lactosylceramide-alpha-2,3-sialyltransferase (ST3Gal5) leads to a severe neuropathology, but in ST3Gal5 knock-out ( St3gal5-/- ) mice the absence of GM3 and associated a-, b- and c-series gangliosides is partially compensated by 0-series gangliosides and there is no overt behavioural phenotype. Here, we sought to examine the behavioural and molecular consequences of GM3 loss more closely. Mutants of both sexes exhibited impaired conditioned taste aversion in an inhibitory learning task and anxiety-like behaviours in the open field, moderate motor deficits, abnormal social interactions, excessive grooming and rearing behaviours. Taken together, the aberrant behaviours are suggestive of an autism spectrum disorder (ASD)-like syndrome. Molecular analysis showed decreased gene and protein expression of proteolipid protein-1 ( Plp1 ) and over expression of proinflammatory cytokines, which has been associated with ASD-like syndromes. The inflammatory and behavioural responses to lipopolysaccharide (LPS) were also altered in the St3gal5-/- mice compared to wild-type, which is indicative of the importance of GM3 gangliosides in regulating immune responses. Together, the St3gal5-/- mice display ASD-like behavioural features, altered response to systemic inflammation, signs of hypomyelination and neuroinflammation, which suggests that deficiency in a- and b-series gangliosides could contribute to the development of an ASD-like pathology in humans., Competing Interests: On behalf of all authors, I would like to state that none of the authors involved in the work have any competing interest., (© 2021 The Authors.)

The central nervous system (CNS) is highly vascularized where neuronal cells are located in proximity to endothelial cells, astroglial limitans, and neuronal processes constituting integrated neurovascular units. In contrast to many other organs, the CNS has a blood-brain barrier (BBB), which becomes compromised due to infection, neuroinflammation, neurodegeneration, traumatic brain injury, and other reasons. BBB disruption is presumably involved in neuronal injury during epilepsy and psychiatric disorders. Therefore, many types of neuropsychological disorders are accompanied by an increase in BBB permeability leading to direct contact of circulating blood cells in the capillaries with neuronal cells in the CNS. The second most abundant type of blood cells are platelets, which come after erythrocytes and outnumber ~100-fold circulating leukocytes. When BBB becomes compromised, platelets swiftly respond to the vascular injury and become engaged in thrombosis and hemostasis. However, more recent studies demonstrated that platelets could also enter CNS parenchyma and directly interact with neuronal cells. Within CNS, platelets become activated by recognizing major brain gangliosides on the surface of astrocytes and neurons and releasing a milieu of pro-inflammatory mediators, neurotrophic factors, and neurotransmitters. Platelet-derived factors directly stimulate neuronal electric and synaptic activity and promote the formation of new synapses and axonal regrowth near the site of damage. Despite such active involvement in response to CNS damage, the role of platelets in neurological disorders was not extensively studied, which will be the focus of this review., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kopeikina and Ponomarev.)

Key Points: Alzheimer's disease (AD) patients and transgenic mice have beta-amyloid (Aβ) aggregation in the gastrointestinal (GI) tract. It is possible that Aβ from the periphery contributes to the load of Aβ in the brain, as Aβ has prion-like properties. The present investigations demonstrate that Aβ injected into the GI tract of ICR mice is internalised into enteric cholinergic neurons; at 1 month, administration of Aβ into the body of the stomach and the proximal colon was observed to partly redistribute to the fundus and jejunum; at 1 year, vagal and cerebral β-amyloidosis was present, and mice exhibited GI dysfunction and cognitive deficits. These data reveal a previously undiscovered mechanism that potentially contributes to the development of AD., Abstract: Alzheimer's disease (AD) is the most common age-related cause of dementia, characterised by extracellular beta-amyloid (Aβ) plaques and intracellular phosphorylated tau tangles in the brain. Aβ deposits have also been observed in the gastrointestinal (GI) tract of AD patients and transgenic mice, with overexpression of amyloid precursor protein. In the present studies, we investigate whether intra-GI administration of Aβ can potentially induce amyloidosis in the central nervous system (CNS) and AD-related pathology such as dementia. We micro-injected Aβ1-42 oligomers (4 μg per site, five sites) or vehicle (saline, 5 μl) into the gastric wall of ICR mice under general anaesthesia. Immunofluorescence staining and in vivo imaging showed that HiLyte Fluor 555-labelled Aβ1-42 had migrated within 3 h via the submucosa to nearby areas and was internalised into cholinergic neurons. At 1 month, HiLyte Fluor 555-labelled Aβ1-42 in the body of the stomach and proximal colon had partly re-distributed to the fundus and jejunum. At 1 year, the jejunum showed functional alterations in neuromuscular coupling (P < 0.001), and Aβ deposits were present in the vagus and brain, with animals exhibiting cognitive impairments in the Y-maze spontaneous alteration test (P < 0.001) and the novel object recognition test (P < 0.001). We found that enteric Aβ oligomers induce an alteration in gastric function, amyloidosis in the CNS, and AD-like dementia via vagal mechanisms. Our results suggest that Aβ load is likely to occur initially in the GI tract and may translocate to the brain, opening the possibility of new strategies for the early diagnosis and prevention of AD., (© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The modern lifestyle is associated with exposure to "psychological" or "emotional" stress. A growing portion of the population is exposed to emotional stress that results in a high incidence of anxiety disorders, a serious social problem. With this rise, there is a need for understanding the neurobiological causes of stress-induced anxiety and to offer safe remedies for this condition. Side effects of existing pharmaceuticals necessitate the search for alternatives. Having fewer adverse effects than classic remedies, natural extract-based therapies can be a promising solution. Here, we applied a model of emotional stress in BALB/c mice using ultrasound exposure to evoke the signs of anxiety-like behavior. We examined the behavioral and molecular impact of ultrasound and administration of herbal antioxidant/anti-inflammatory treatment (HAT) on AMPA receptor expression, markers of plasticity, inflammation and oxidative stress. A 3-week ultrasound exposure increased scores of anxiety-like behaviors in the standard tests and altered hippocampal expression as well as internalization of AMPA receptor subunits GluA1-A3. Concomitant treatment with HAT has prevented increases of anxiety-like behaviors and other behavioral changes, normalized hippocampal malondialdehyde content, GSK3β and pro-inflammatory cytokines Il-1β and Il-6, and the number of Ki67-positive cells. Levels of malondialdehyde, a common measure of oxidative stress, significantly correlated with the investigated end-points in stressed, but not in non-stressed animals. Our results emphasize the role of oxidative stress in neurobiological abnormalities associated with experimentally induced condition mimicking emotional stress in rodents and highlight the potential therapeutic use of anti-oxidants like herbal compositions for management of stress-related emotional disturbances within the community.

Background: In Alzheimer's diseases, beta-amyloid may act as prion-like protein and migrate from the gastrointestinal tract towards the brain. Soy flavonoids have been identified as neuroprotective against cognitive loss in human. Diet with soy flavonoids may be used to slow down the progression of Alzheimer's diseases., Methods and Results: We performed in-vitro tissue culture experiments using myenteric plexus longitudinal muscle layers isolated from the ileum and colon of ICR mice. Beta-amyloid can be taken up into myenteric neurons and induce neuron degeneration, which is protected by flavonoids compounds, including daidzein, genistein, glycitein and luteolin. We also administered oligomeric beta-amyloid (1-42) (total dose: 8 μg) into the gastrointestinal walls of ICR mice and conducted memory tests and gastrointestinal function assessments after 6 and 12 months. Mice treated with beta-amyloid exhibited minor learning deficits in a T-maze memory test at 6 months and significant memory impairment in a novel object recognition task at 12 months. These impairments were prevented by soy flavonoids. Tracking studies performed using fluorescently tagged beta-amyloid found that, beta-amyloid injected at the stomach can aggregate within the layer of myenteric neurons and migrate to the jejunum or via the vagus nerves to the brain after 1 month. Reductions in the gastrointestinal tissue weight and the spontaneous ileal contraction frequency were also observed at 6 and 12 months, respectively., Conclusion: Our findings indicate that beta-amyloid can migrate from the gastrointestinal tract to the brain to induce cognitive impairments. Furthermore, chronic soy flavonoids in drinking water have protective actions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The drugs currently available for treating epilepsy are only partially effective in managing this condition. Therefore, it is crucial to investigate new pathways that induce and promote epilepsy development. Previously, we found that platelets interact with neuronal glycolipids and actively secrete pro-inflammatory mediators during central nervous system (CNS) pathological conditions such as neuroinflammation and traumatic brain injury (TBI). These factors increase the permeability of the blood-brain barrier (BBB), which may create a predisposition to epileptic seizures. In this study, we demonstrated that platelets substantially enhanced epileptic seizures in a mouse model of pentylenetetrazole (PTZ) -induced seizures. We found that platelets actively secreted serotonin, contributed to increased BBB permeability, and were present in the CNS parenchyma during epileptic seizures. Furthermore, platelets directly stimulated neuronal electric activity and induced the expression of specific genes related to early neuronal response, neuroinflammation, and oxidative phosphorylation, leading to oxidative stress in neurons. The intracranial injection of physiological numbers of platelets that mimicked TBI-associated bleeding was sufficient to induce severe seizures, which resembled conventional PTZ-induced epileptic activity. These findings highlight a conceptually new role of platelets in the development of epileptic seizures, and indicate a potential new therapeutic approach targeting platelets to prevent and treat epilepsy., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Twenty years ago, the scientific community exhibited relatively little interest in the study of microglial cells. However, recent technical and conceptual advances in this field have greatly increased interest in the basic biology of these cells within various neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease, and traumatic brain/spinal cord injuries. The main functions of these cells in the normal central nervous system (CNS) remain poorly understood, despite considerable elucidation of their roles in pathological conditions. Microglia populate the brain before birth and remain in close lifelong contact with CNS-resident cells under the influence of the local microenvironment. Within the CNS parenchyma, microglia actively interact with two main cell types, astrocytes and neurons, which produce many factors that affect microglia phenotypes in the normal CNS and during neuroinflammation. These factors include interleukin (IL)-34, macrophage colony-stimulating factor, transforming growth factor-β, and IL-4, which promote microglial expansion, survival, and differentiation to an anti-inflammatory phenotype in the normal CNS. Under inflammatory conditions, however, astrocytes produce several pro-inflammatory factors that contribute to microglial activation. The interactions of microglia with neurons in the normal and diseased CNS are especially intriguing. Microglia are known to interact actively with neurons by facilitating axonal pruning during development, while neurons provide specific factors that alter microglial phenotypes and functions. This review focuses mainly on the roles of soluble neuronal factors that affect microglial phenotypes and functions and the possible involvement of these factors in the pathology of neurodegenerative diseases., (Copyright © 2019 Veremeyko, Yung, Dukhinova, Strekalova and Ponomarev.)

The negative societal impacts associated with the increasing prevalence of violence and aggression is increasing, and, with this rise, is the need to understand the molecular and cellular changes that underpin ultrasound-induced aggressive behavior. In mice, stress-induced aggression is known to alter AMPA receptor subunit expression, plasticity markers, and oxidative stress within the brain. Here, we induced aggression in BALB/c mice using chronic ultrasound exposure and examined the impact of the psychoactive anti-oxidant compounds thiamine (vitamin B1), and its derivative benfotiamine, on AMPA receptor subunit expression, established plasticity markers, and oxidative stress. The administration of thiamine or benfotiamine (200 mg/kg/day) in drinking water decreased aggressive behavior following 3-weeks of ultrasound exposure and benfotiamine, reduced floating behavior in the swim test. The vehicle-treated ultrasound-exposed mice exhibited increases in protein carbonyl and total glutathione, altered AMPA receptor subunits expression, and decreased expression of plasticity markers. These ultrasound-induced effects were ameliorated by thiamine and benfotiamine treatment; in particular both antioxidants were able to reverse ultrasound-induced changes in GluA1 and GluA2 subunit expression, and, within the prefrontal cortex, significantly reversed the changes in protein carbonyl and polysialylated form of neural cell adhesion molecule (PSA-NCAM) expression levels. Benfotiamine was usually more efficacious than thiamine. Thus, the thiamine compounds were able to counteract ultrasound-induced aggression, which was accompanied by the normalization of markers that have been showed to be associated with ultrasound-induced aggression. These commonly used, orally-active compounds may have considerable potential for use in the control of aggression within the community. This article is part of the Special Issue entitled 'Current status of the neurobiology of aggression and impulsivity'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although it was suggested that gangliosides play an important role in the binding of amyloid fragments to neuronal cells, the exact role of gangliosides in Alzheimer's disease (AD) pathology remains unclear. To understand the role of gangliosides in AD pathology in vivo, we crossed st3gal5-deficient (ST3 -/- ) mice that lack major brain gangliosides GM1, GD1a, GD3, GT1b, and GQ1b with 5XFAD transgenic mice that overexpress 3 mutant human amyloid proteins AP695 and 2 presenilin PS1 genes. We found that ST3 -/- 5XFAD mice have a significantly reduced burden of amyloid depositions, low level of neuroinflammation, and did not exhibit neuronal loss or synaptic dysfunction. ST3 -/- 5XFAD mice performed significantly better in a cognitive test than wild-type (WT) 5XFAD mice, which was comparable with WT nontransgenic mice. Treatment of WT 5XFAD mice with the sialic acid-specific Limax flavus agglutinin resulted in substantial improvement of AD pathology to a level of ST3 -/- 5XFAD mice. Thus, our findings highlight an important role for gangliosides as a target for the treatment of AD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Emotional stress is a form of stress evoked by processing negative mental experience rather than an organic or physical disturbance and is a frequent cause of neuropsychiatric pathologies, including depression. Susceptibility to emotional stress is commonly regarded as a human-specific trait that is challenging to model in other species. Recently, we showed that a 3-week-long exposure to ultrasound of unpredictable alternating frequencies within the ranges of 20-25 kHz and 25-45 kHz can induce depression-like characteristics in laboratory mice and rats. In an anti-depressant sensitive manner, exposure decreases sucrose preference, elevates behavioural despair, increases aggression, and alters serotonin-related gene expression. To further investigate this paradigm, we studied depression/distress-associated markers of neuroinflammation, neuroplasticity, oxidative stress and the activity of glycogen synthase kinase-3 (GSK-3) isoforms in the hippocampus of male mice. Stressed mice exhibited a decreased density of Ki67-positive and DCX-positive cells in the subgranular zone of hippocampus, and altered expression of brain-derived neurotrophic factor (BDNF), its receptor TrkB, and anti-apoptotic protein kinase B phosphorylated at serine 473 (AktpSer473). The mice also exhibited increased densities of Iba-1-positive cells, increased oxidative stress, increased levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) in the hippocampus and plasma, and elevated activity of GSK-3 isoforms. Together, the results of our investigation have revealed that unpredictable alternating ultrasound evokes behavioural and molecular changes that are characteristic of the depressive syndrome and validates this new and simple method of modeling emotional stress in rodents., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In contrast to peripheral macrophages, microglia in the central nervous system (CNS) exhibit a specific deactivated phenotype; however, it is not clear how this phenotype is maintained. Two alternative hypotheses were postulated recently: (a) microglia differ from peripheral macrophages being derived from the yolk sac (YS), whereas peripheral macrophages originate from bone marrow (BM); (b) microglia acquire a specific phenotype under the influence of the CNS microenvironment. We have previously shown that microglia express miR-124, which was also induced in BM-derived macrophages co-cultured with a neurons. We here investigated the possibility of horizontal transfer of the neuron-specific microRNAs miR-124 and miR-9 from primary neurons to microglia/macrophages. We found that after incubation with neuronal conditioned media (NCM), macrophages downregulated activation markers MHC class II and CD45. Neither cultured adult microglia nor YS- and BM-derived macrophages demonstrated intrinsic levels of miR-124 expression. However, after incubation with NCM, miR-124 was induced in both YS- and BM-derived macrophages. Biochemical analysis demonstrated that the NCM contained miR-124 and miR-9 in complex with small proteins, large high-density lipoproteins (HDLs), and exosomes. MiR-124 and miR-9 were promptly released from neurons, and this process was inhibited by tetrodotoxin, indicating an important role of neuronal electric activity in secretion of these microRNAs. Incubation of macrophages with exogenous miR-124 resulted in efficient translocation of miR-124 into the cytoplasm. This study demonstrates an important role of neuronal miRNAs in communication of neurons with microglia, which favors the hypothesis that microglia acquire a specific phenotype under the influence of the CNS microenvironment., (© 2018 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.)

Monoclonal antibodies (mAbs) are an important class of therapeutic agents approved for the therapy of many types of malignancies. However, in certain cases applications of conventional mAbs have several limitations in anticancer immunotherapy. These limitations include insufficient efficacy and adverse effects. The antigen-binding fragments of antibodies have a considerable potential to overcome the disadvantages of conventional mAbs, such as poor penetration into solid tumors and Fc-mediated bystander activation of the immune system. Fragments of antibodies retain antigen specificity and part of functional properties of conventional mAbs and at the same time have much better penetration into the tumors and a greatly reduced level of adverse effects. Recent advantages in antibody engineering allowed to produce different types of antibody fragments with improved structure and properties for efficient elimination of tumor cells. These molecules opened up new perspectives for anticancer therapy. Here, we will overview the structural features of the various types of antibody fragments and their applications for anticancer therapy as separate molecules and as part of complex conjugates or structures. Mechanisms of antitumor action of antibody fragments as well as their advantages and disadvantages for clinical application will be discussed in this review., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

[This corrects the article DOI: 10.3389/fimmu.2018.02515.].

It is generally accepted that inflammation within the CNS contributes to neurodegeneration after traumatic brain injury (TBI), but it is not clear how inflammation is initiated in the absence of infection and whether this neuroinflammation is predominantly beneficial or detrimental. We have previously found that brain-enriched glycosphingolipids within neuronal lipid rafts (NLR) induced platelet degranulation and secretion of neurotransmitters and pro-inflammatory factors. In the present study, we compared TBI-induced inflammation and neurodegeneration in wild-type vs. St3gal5 deficient (ST3 -/- ) mice that lack major CNS-specific glycosphingolipids. After TBI, microglial activation and CNS macrophage infiltration were substantially reduced in ST3 -/- animals. However, ST3 -/- mice had a larger area of CNS damage with marked neuronal/axonal loss. The interaction of platelets with NLR stimulated neurite growth, increased the number of PSD95-positive dendritic spines, and intensified neuronal activity. Adoptive transfer and blocking experiments provide further that platelet-derived serotonin and platelet activating factor plays a key role in the regulation of sterile neuroinflammation, hemorrhage and neuronal plasticity after TBI., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The process of macrophage polarization is involved in many pathologies such as anti-cancer immunity and autoimmune diseases. Polarized macrophages exhibit various levels of plasticity when M2/M(IL-4) macrophages are reprogrammed into an M1-like phenotype following treatment with IFNγ and/or LPS. At the same time, M1 macrophages are resistant to reprogramming in the presence of M2-like stimuli. The molecular mechanisms responsible for the macrophages polarization, plasticity of M2 macrophages, and lack of plasticity in M1 macrophages remain unknown. Here, we explored the role of Egr2 in the induction and maintenance of macrophage M1 and M2 polarization in the mouse in vitro and in vivo models of inflammation. Egr2 knockdown with siRNA treatment fail to upregulate either M1 or M2 markers upon stimulation, and the overexpression of Egr2 potentiated M1 or M2 marker expression following polarization. Polarisation with M2-like stimuli (IL-4 or IL-13) results in increased Egr2 expression, but macrophages stimulated with M1-like stimuli (IFNγ, LPS, IL-6, or TNF) exhibit a decrease in Egr2 expression. Egr2 was critical for the expression of transcription factors CEBPβ and PPARγ in M2 macrophages, and CEBPβ was highly expressed in M1-polarized macrophages. In siRNA knockdown studies the transcription factor CEBPβ was found to negatively regulate Egr2 expression and is likely to be responsible for the maintenance of the M1-like phenotype and lack plasticity. During thioglycolate-induced peritonitis, adoptively transferred macrophages with Egr2 knockdown failed to become activated as determined by upregulation of MHC class II and CD86. Thus, our study indicates that Egr2 expression is associated with the ability of unstimulated or M2 macrophages to respond to stimulation with inflammatory stimuli, while low levels of Egr2 expression is associated with non-responsiveness of macrophages to their activation.

While the insulin receptor (IR) was found in the CNS decades ago, the brain was long considered to be an insulin-insensitive organ. This view is currently revisited, given emerging evidence of critical roles of IR-mediated signaling in development, neuroprotection, metabolism, and plasticity in the brain. These diverse cellular and physiological IR activities are distinct from metabolic IR functions in peripheral tissues, thus highlighting region specificity of IR properties. This particularly concerns the fact that two IR isoforms, A and B, are predominantly expressed in either the brain or peripheral tissues, respectively, and neurons express exclusively IR-A. Intriguingly, in comparison with IR-B, IR-A displays high binding affinity and is also activated by low concentrations of insulin-like growth factor-2 (IGF-2), a regulator of neuronal plasticity, whose dysregulation is associated with neuropathologic processes. Deficiencies in IR activation, insulin availability, and downstream IR-related mechanisms may result in aberrant IR-mediated functions and, subsequently, a broad range of brain disorders, including neurodevelopmental syndromes, neoplasms, neurodegenerative conditions, and depression. Here, we discuss findings on the brain-specific features of IR-mediated signaling with focus on mechanisms of primary receptor activation and their roles in the neuropathology. We aimed to uncover the remaining gaps in current knowledge on IR physiology and highlight new therapies targeting IR, such as IR sensitizers., (© 2018 John Wiley & Sons Ltd.)

Recent studies suggest that in addition to their common function in the regulation of thrombosis and hemostasis, platelets also contribute to tissue inflammation affecting adaptive immunity. Platelets have a number of pro-inflammatory and regulatory mediators stored in their α-granules and dense granules, which are promptly released at sites of inflammation or tissue injury. Platelet-derived mediators include cytokines (IL-1α, IL-1β, and TGFβ1), chemokines (CXCL4 and CCL3), immunomodulatory neurotransmitters (serotonin, dopamine, epinephrine, histamine, and GABA), and other low-molecular-weight mediators. In addition, activated platelets synthesize a number of lipid pro-inflammatory mediators such as platelet-activating factor and prostaglandins/thromboxanes. Notably, platelets express multiple toll-like receptors and MHC class I on their surface and store IgG in their α-granules. Platelet-derived factors are highly effective in directly or indirectly modulating the priming and effector function of various subsets of T cells. Besides secreting soluble factors, activated platelets upregulate a number of integrins, adhesion molecules, and lectins, leading to the formation of platelet-T cells aggregates. Activated platelets are able to instantly release neurotransmitters acting similar to neuronal presynaptic terminals, affecting CD4 T cells and other cells in close contact with them. The formation of platelet-T cell aggregates modulates the functions of T cells via direct cell-cell contact interactions and the local release of soluble factors including neurotransmitters. New data suggest an important role for platelets as neuronal and innate-like cells that directly recognize damage- or pathogen- associated molecular patterns and instantly communicate with T cells.

Although it has been demonstrated that cAMP pathway affect both adaptive and innate cell functions, the role of this pathway in the regulation of T-cell-mediated central nervous system (CNS) autoimmune inflammation, such as in experimental autoimmune encephalomyelitis (EAE), remains unclear. It is also unclear how cAMP pathway affects the function of CD4 T cells in vivo at the site of inflammation. We found that adenylyl cyclase activator Forskolin besides inhibition of functions autoimmune CD4 T cells also upregulated microRNA (miR)-124 in the CNS during EAE, which is associated with M2 phenotype of microglia/macrophages. Our study further established that in addition to direct influence of cAMP pathway on CD4 T cells, stimulation of this pathway promoted macrophage polarization toward M2 leading to indirect inhibition of function of T cells in the CNS. We demonstrated that Forskolin together with IL-4 or with Forskolin together with IL-4 and IFNγ effectively stimulated M2 phenotype of macrophages indicating high potency of this pathway in reprogramming of macrophage polarization in Th2- and even in Th1/Th2-mixed inflammatory conditions such as EAE. Mechanistically, Forskolin and/or IL-4 activated ERK pathway in macrophages resulting in the upregulation of M2-associated molecules miR-124, arginase (Arg)1, and Mannose receptor C-type 1 (Mrc1), which was reversed by ERK inhibitors. Administration of Forskolin after the onset of EAE substantially upregulated M2 markers Arg1, Mrc1, Fizz1, and Ym1 and inhibited M1 markers nitric oxide synthetase 2 and CD86 in the CNS during EAE resulting in decrease in macrophage/microglia activation, lymphocyte and CD4 T cell infiltration, and the recovery from the disease. Forskolin inhibited proliferation and IFNγ production by CD4 T cells in the CNS but had rather weak direct effect on proliferation of autoimmune T cells in the periphery and in vitro , suggesting prevalence of indirect effect of Forskolin on differentiation and functions of autoimmune CD4 T cells in vivo . Thus, our data indicate that Forskolin has potency to skew balance toward M2 affecting ERK pathway in macrophages and indirectly inhibit pathogenic CD4 T cells in the CNS leading to the suppression of autoimmune inflammation. These data may have also implications for future therapeutic approaches to inhibit autoimmune Th1 cells at the site of tissue inflammation.

Neuronal cells are probably the less studied cells regarding their heterogeneity on a single cell or population levels. One of the main problems of studying of individual neurons is the presence of long processes (axons) on differentiated adult neurons that hamper their isolation without significant damage to the cells. Therefore, the most common method to study neuronal cells is immunofluorescent microscopy of sections of the brain, which remains poorly quantitative and allows analyzing a small number of fixed cells. Also, immunofluorescent microscopy has a number of staining artifacts since histology section has high level of autofluorescence and non-specific binding of fluorescent probes. Alternative methods that could overcome disadvantages of immunofluorescent histology include flow cytometry, scanning cytometry, and laser interferometry. Flow cytometry and, to some extent of degree, scanning cytometry allow performing analysis of multiple markers with a low level of non-specific background and very robust statistics. Laser interferometry allows studies intact, alive neurons without staining. Limitations and advantages of these methods are discussed in this chapter.

The resident macrophages of the central nervous system (CNS), also known as microglia, and blood-derived macrophages play an important role in the functional activity of the normal CNS, as well as in the development of neuroinflammation during various neurodegenerative disorders. Microglia and macrophages represent heterogeneous populations, which can modulate CNS environment and have different effects on neuronal regeneration. In this chapter, the main features of microglial and macrophage subsets and current methods for investigation of their heterogeneity will be discussed.

Extracellular vesicle (EV) production is a universal feature of metazoan cells as well as prokaryotes (bMVs - bacterial microvesicles). They are small vesicles with phospholipid membrane carrying proteins, DNA and different classes of RNAs and are heavily involved in intercellular communication acting as vectors of information to target cells. For the last decade, the interest in EV research has exponentially increased though thorough studies of their roles in various pathologies that was not previously possible due to technical limitations. This review focuses on research evaluating the role of EV production in gastrointestinal (GI) cancer development in conjunction with GI microbiota and inflammatory diseases. We also discuss recent studies on the promising role of EVs and their content as biomarkers for early diagnosis of GI cancers. The bMVs have also been implicated in the pathogenesis of GI chronic inflammatory diseases, however, possible role of bMVs in tumorigenesis remains underestimated. We propose that EVs from eukaryotic cells as well as from different microbial, fungi, parasitic species and edible plants in GI tract act as mediators of intracellular and inter-species communication, particularly facilitating tumor cell survival and multi-drug resistance. In conclusion, we suggest that matching sequences from EV proteomes (available from public databases) with known protein sequences of microbiome gut bacteria will be useful in identification of antigen mimicry between evolutionary conservative protein sequences. Using this approach we identified Bacteroides spp. pseudokinase with activation loop and homology to PDGFRα, providing a proof-of-concept strategy. We speculate that existence of microbial pseudokinase that 'mimics' PDGFRα may be related to PDGFRα and Bacteroides spp. roles in colorectal carcinogenesis that require further investigation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Rationale: Platelets are known to participate in vascular pathologies; however, their role in neuroinflammatory diseases, such as multiple sclerosis (MS), is unknown. Autoimmune CD4 T cells have been the main focus of studies of MS, although the factors that regulate T-cell differentiation toward pathogenic T helper-1/T helper-17 phenotypes are not completely understood., Objective: We investigated the role of platelets in the modulation of CD4 T-cell functions in patients with MS and in mice with experimental autoimmune encephalitis, an animal model for MS., Methods and Results: We found that early in MS and experimental autoimmune encephalitis, platelets degranulated and produced soluble factors serotonin (5-hydroxytryptamine), platelet factor 4, and platelet-activating factor, which specifically stimulated differentiation of T cells toward pathogenic T helper-1, T helper-17, and interferon-γ/interleukin-17-producing CD4 T cells. At the later stages of MS and experimental autoimmune encephalitis, platelets became exhausted in their ability to produce proinflammatory factors and stimulate CD4 T cells but substantially increased their ability to form aggregates with CD4 T cells. Formation of platelet-CD4 T-cell aggregates involved the interaction of CD62P on activated platelets with adhesion molecule CD166 on activated CD4 T cells, contributing to downmodulation of CD4 T-cell activation, proliferation, and production of interferon-γ. Blocking of formation of platelet-CD4 T-cell aggregates during progression of experimental autoimmune encephalitis substantially enhanced proliferation of CD4 T cells in the central nervous system and the periphery leading to exacerbation of the disease., Conclusion: Our study indicates differential roles for platelets in the regulation of functions of pathogenic CD4 T cells during initiation and progression of central nervous system autoimmune inflammation., (© 2015 American Heart Association, Inc.)

Background: Glatiramer acetate (GA, Copaxone, Copolymer-1) is an FDA approved drug for the treatment of MS and it is very effective in suppressing neuroinflammation in experimental autoimmune encephalitis (EAE), an animal model of MS. Although this drug was designed to inhibit pathogenic T cells, the exact mechanism of EAE/MS suppression by GA is still not well understood. Previously we presented evidence that platelets become activated and promote neuroinflammation in EAE, suggesting a possible pathogenic role of platelets in MS and EAE. We hypothesized that GA could inhibit neuroinflammation by affecting not only immune cells but also platelets., Methodology/principal Findings: We investigated the effect of GA on the activation of human platelets in vitro: calcium influx, platelet aggregation and expression of activation markers. Our results in human platelets were confirmed by in-vitro and in-vivo studies of modulation of functions of platelets in mouse model. We found that GA inhibited thrombin-induced calcium influx in human and mouse platelets. GA also decreased thrombin-induced CD31, CD62P, CD63, and active form of αIIbβ3 integrin surface expression and formation of platelet aggregates for both mouse and human platelets, and prolonged the bleeding time in mice by 2.7-fold. In addition, we found that GA decreased the extent of macrophage activation induced by co-culture of macrophages with platelets., Conclusions: GA inhibited the activation of platelets, which suggests a new mechanism of GA action in suppression of EAE/MS by targeting platelets and possibly preventing their interaction with immune cells such as macrophages. Furthermore, the reduction in platelet activation by GA may have additional cardiovascular benefits to prevent thrombosis.

Background: Ganglioside GD2 is expressed on plasma membranes of various types of malignant cells. One of the most promising approaches for cancer immunotherapy is the treatment with monoclonal antibodies recognizing tumor-associated markers such as ganglioside GD2. It is considered that major mechanisms of anticancer activity of anti-GD2 antibodies are complement-dependent cytotoxicity and/or antibody-mediated cellular cytotoxicity. At the same time, several studies suggested that anti-GD2 antibodies are capable of direct induction of cell death of number of tumor cell lines, but it has not been investigated in details. In this study we investigated the functional role of ganglioside GD2 in the induction of cell death of multiple tumor cell lines by using GD2-specific monoclonal antibodies., Methods: Expression of GD2 on different tumor cell lines was analyzed by flow cytometry using anti-GD2 antibodies. By using HPTLC followed by densitometric analysis we measured the amount of ganglioside GD2 in total ganglioside fractions isolated from tumor cell lines. An MTT assay was performed to assess viability of GD2-positive and -negative tumor cell lines treated with anti-GD2 mAbs. Cross-reactivity of anti-GD2 mAbs with other gangliosides or other surface molecules was investigated by ELISA and flow cytometry. Inhibition of GD2 expression was achieved by using of inhibitor for ganglioside synthesis PDMP and/or siRNA for GM2/GD2 and GD3 synthases., Results: Anti-GD2 mAbs effectively induced non-classical cell death that combined features of both apoptosis and necrosis in GD2-positive tumor cells and did not affect GD2-negative tumors. Anti-GD2 mAbs directly induced cell death, which included alteration of mitochondrial membrane potential, induction of apoptotic volume decrease and cell membrane permeability. This cytotoxic effect was mediated exclusively by specific binding of anti-GD2 antibodies with ganglioside GD2 but not with other molecules. Moreover, the level of GD2 expression correlated with susceptibility of tumor cell lines to cytotoxic effect of anti-GD2 antibodies., Conclusions: Results of this study demonstrate that anti-GD2 antibodies not only passively bind to the surface of tumor cells but also directly induce rapid cell death after the incubation with GD2-positive tumor cells. These results suggest a new role of GD2 as a receptor that actively transduces death signal in malignant cells.

High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE(+) cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations.

Monocytic cells exhibit a high level of heterogeneity and have two distinct modes of their activation: 1) classical M1 path associated with inflammation and tissue damage, and 2) alternative M2 path. Although it has been demonstrated that M2 macrophages play an important role in the regulation of the allergic immune responses, tissue maintenance and repair, little is known about the mechanisms that determine the M2 phenotype. We have previously shown that miR-124 is expressed in microglia that exhibit the M2 phenotype and overexpression of miR-124 in macrophages resulted in downregulation of a number of M1 markers (MHC class II, CD86) and up-regulation of several M2 markers (Fizz1, Arg1). We further investigated whether the polarization of macrophages towards the M2 phenotype induced miR-124 expression. We found that exposure of cells to IL-4 and IL-13 resulted in the upregulation of miR-124 in macrophages. We also demonstrated that IL-4 induced expression of three miR-124 precursor transcripts with predominant expression of pri-miR-124.3, suggesting regulation of miR-124 expression by IL-4 on a transcriptional level. Expression of miR-124 in microglia did not depend on IL-4 and/or IL-13, whereas expression of miR-124 in lung resident macrophages was IL-4 and IL-13-dependent and was upregulated by systemic administration of IL-4 or during allergic inflammation. Upregulation of several M2 markers (CD206, Ym1) and downregulation of the M1 markers (CD86, iNOS, TNF) in M2-polarized macrophages was abrogated by a miR-124 inhibitor, suggesting that this microRNA contributed to the M2 phenotype development and maintenance. Finally we showed that human CD14(+)CD16(+) intermediate monocytes, which are found in increased numbers in patients with allergies and bronchial asthma, expressed high levels of miR-124 and exhibited other properties of M2-like cells. Thus, our study suggests that miR-124 serves as a regulator of the M2 polarization in various subsets of monocytic cells both in vitro and in vivo.

Background: The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance., Results: MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform., Conclusions: Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes.

Platelets respond to vascular damage and contribute to inflammation, but their role in the neurodegenerative diseases is unknown. We found that the systemic administration of brain lipid rafts induced a massive platelet activation and degranulation resulting in a life-threatening anaphylactic-like response in mice. Platelets were engaged by the sialated glycosphingolipids (gangliosides) integrated in the rigid structures of astroglial and neuronal lipid rafts. The brain-abundant gangliosides GT1b and GQ1b were specifically recognized by the platelets and this recognition involved multiple receptors with P-selectin (CD62P) playing the central role. During the neuroinflammation, platelets accumulated in the central nervous system parenchyma, acquired an activated phenotype and secreted proinflammatory factors, thereby triggering immune response cascades. This study determines a new role of platelets which directly recognize a neuronal damage and communicate with the cells of the immune system in the pathogenesis of neurodegenerative diseases.

MicroRNAs (miRNAs) are a class of small (∼22 nucleotides) noncoding RNAs involved in the regulation of gene expression at the post-translational level. It is estimated that 30-90% of human genes are regulated by miRNAs, which makes these molecules of great importance for cell growth, activation, and differentiation. Microglia is CNS-resident cells of a myeloid lineage that play an important role in immune surveillance and are actively involved in many neurologic pathologies. Although the exact origin of microglia remains enigmatic, it is established that primitive macrophages from a yolk sac populate the brain and spinal cord in normal conditions throughout development. During various pathological events such as neuroinflammation, bone marrow derived myeloid cells also migrate into the CNS. Within the CNS, both primitive macrophages from the yolk sac and bone marrow derived myeloid cells acquire a specific phenotype upon interaction with other cell types within the CNS microenvironment. The factors that drive differentiation of progenitors into microglia and control the state of activation of microglia and bone marrow-derived myeloid cells within the CNS are not well understood. In this review we will summarize the role of miRNAs during activation and differentiation of myeloid cells. The role of miR-124 in the adaptation of microglia and macrophages to the CNS microenvironment will be further discussed. We will also summarize the role of miRNAs as modulators of activation of microglia and microphages. Finally, we will describe the role of miR-155 and miR-124 in the polarization of macrophages towards classically and alternatively activated phenotypes., (Copyright © 2012 Wiley Periodicals, Inc.)

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)(1). These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)(2). Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers(3). The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation(4) and pathologies such as inflammation(5). MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages(6) and microglia(7). In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Background: MicroRNAs (miRNAs) are regulatory molecules that play an important role in many physiological processes, including cell growth, differentiation, and apoptosis. In addition to modulating normal cellular functions, it has also been reported that miRNAs are involved in the development of many pathologies, including cardiovascular diseases, cancer, inflammation, and neurodegeneration. Methods for the sensitive detection and measurement of specific miRNAs and their cellular targets are essential for both basic research endeavours, as well as diagnostic efforts aimed at understanding the role of miRNAs in disease processes., Findings: In this study, we describe a novel, imaging cytometry-based protocol that allows for simultaneous visualisation and quantification of miRNAs and their putative targets. We validated this methodology in a neuronal cell line by examining the relationship of the miRNA miR-124 and its known target, cyclin dependent kinase 6 (CDK6). We found that ectopic overexpression of miR-124 resulted in the downregulation of CDK6, decreased cellular proliferation, and induced cellular morphological changes., Conclusions: This method is suitable for analysing the expression and cellular localisation of miRNAs and target proteins in small cell subsets within a heterogeneous cell suspension. We believe that our cytometry-based methodology will be easily adaptable to miRNA studies in many areas of biomedical research including neuroscience, stem cell biology, immunology, and oncology.

MicroRNAs are a family of regulatory molecules involved in many physiological processes, including differentiation and activation of cells of the immune system. We found that brain-specific miR-124 is expressed in microglia but not in peripheral monocytes or macrophages. When overexpressed in macrophages, miR-124 directly inhibited the transcription factor CCAAT/enhancer-binding protein-α (C/EBP-α) and its downstream target PU.1, resulting in transformation of these cells from an activated phenotype into a quiescent CD45(low), major histocompatibility complex (MHC) class II(low) phenotype resembling resting microglia. During experimental autoimmune encephalomyelitis (EAE), miR-124 was downregulated in activated microglia. Peripheral administration of miR-124 in EAE caused systemic deactivation of macrophages, reduced activation of myelin-specific T cells and marked suppression of disease. Conversely, knockdown of miR-124 in microglia and macrophages resulted in activation of these cells in vitro and in vivo. These findings identify miR-124 both as a key regulator of microglia quiescence in the central nervous system and as a previously unknown modulator of monocyte and macrophage activation.

Macrophages are thought to play an important role in the maintenance of immune privilege in the testis, which functions to prevent immune responses to developing sperm. Two populations of macrophages are known to exist in the testis, one of which exhibits immunosuppressive activity. Macrophages that are alternatively activated with either IL-4 or IL-13 have been shown to be anti-inflammatory and promote wound healing. Expression of the Ym1 protein is an established marker of alternatively activated macrophages. Testicular macrophages were examined for expression of Ym1 protein, and it was found to be highly expressed in a subpopulation of CD11b(+) cells. Furthermore, we have shown that Ym1 protein expression in the testis is dependent upon IL-13R signaling, and that IL-13 is produced in the testis. These data suggest that IL-13 plays a role in testicular immune privilege by the maintenance of an alternatively activated macrophage population.

Regulation of inflammation in the CNS is essential to prevent irreversible cellular damage that can occur in neurodegenerative diseases such as multiple sclerosis (MS). We investigated the role of interleukin-4 (IL-4) in regulating CNS inflammation using the animal model of MS, experimental autoimmune encephalomyelitis (EAE). We found that CNS-derived IL-4 was a critical regulator because mice with a deficiency in IL-4 production in the CNS, but not the periphery, had exacerbated EAE associated with a significant increase in the absolute number of infiltrating inflammatory cells. We also found that CNS-resident microglial cells in both the resting and activated state produced the protein Ym1, which is a marker of alternatively activated macrophages (aaMphis), in an IL-4-dependent manner. This aaMphi phenotype extended to the lack of nitric oxide (NO) production by activated microglial cells, which is a marker of classically activated macrophages. We also show that IL-4 induced the expression of Ym1 in peripheral infiltrating macrophages, which also produce NO. Thus, macrophages that migrate into the CNS exhibit a dual phenotype. These data indicate that IL-4 production in the CNS is essential for controlling autoimmune inflammation by inducing a microglial cell aaMphi phenotype. Macrophages that have undergone alternative activation have been shown to be important in tissue repair; thus, our results suggest a new role for microglial cells in the regulation of inflammation in the CNS.

The cannabinoid system is immunomodulatory and has been targeted as a treatment for the central nervous system (CNS) autoimmune disease multiple sclerosis. Using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we investigated the role of the CB(1) and CB(2) cannabinoid receptors in regulating CNS autoimmunity. We found that CB(1) receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB(2) receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB(2)-deficient T cells in the CNS during EAE exhibited reduced levels of apoptosis, a higher rate of proliferation and increased production of inflammatory cytokines, resulting in severe clinical disease. Together, our results demonstrate that the cannabinoid system within the CNS plays a critical role in regulating autoimmune inflammation, with the CNS directly suppressing T-cell effector function via the CB(2) receptor.

Multiple sclerosis (MS) is a CNS autoimmune disease believed to be triggered by T cells secreting Th1-specific proinflammatory cytokines, such as GM-CSF. In the animal model of MS, experimental autoimmune encephalomyelitis (EAE), Th1 but not Th2 cells have been shown to induce disease; however, to date, no single encephalitogenic T cell-derived cytokine has been shown to be required for EAE onset. Because GM-CSF-deficient mice have been shown to be resistant to EAE following immunization with myelin self-Ag, we investigated the cellular source of the required GM-CSF and found that GM-CSF production by encephalitogenic T cells, but not CNS resident or other peripheral cells, was required for EAE induction. Furthermore, we showed that microglial cell activation, but not peripheral macrophage activation, was a GM-CSF-dependent process. Activation of microglial cells by the injection of LPS abrogated the GM-CSF requirement for EAE induction, suggesting that microglial cell activation is required for EAE onset. These data also demonstrate that GM-CSF is a critical Th1 cell-derived cytokine required for the initiation of CNS inflammation associated with EAE, and likely MS.

Microglial cells are monocytic lineage cells that reside in the CNS and have the capacity to become activated during various pathological conditions. Although it was demonstrated that activation of microglial cells could be achieved in vitro by the engagement of CD40-CD40L interactions in combination with proinflammatory cytokines, the exact factors that mediate activation of microglial cells in vivo during CNS autoimmunity are ill-defined. To investigate the role of CD40 in microglial cell activation during experimental autoimmune encephalomyelitis (EAE), we used bone marrow chimera mice that allowed us to distinguish microglial cells from peripheral macrophages and render microglial cells deficient in CD40. We found that the first step of microglial cell activation was CD40-independent and occurred during EAE onset. The first step of activation consisted of microglial cell proliferation and up-regulation of the activation markers MHC class II, CD40, and CD86. At the peak of disease, microglial cells underwent a second step of activation, which was characterized by a further enhancement in activation marker expression along with a reduction in proliferation. The second step of microglial cell activation was CD40-dependent and the failure of CD40-deficient microglial cells to achieve a full level of activation during EAE was correlated with reduced expansion of encephalitogenic T cells and leukocyte infiltration in the CNS, and amelioration of clinical symptoms. Thus, our findings demonstrate that CD40 expression on microglial cells is necessary to complete their activation process during EAE, which is important for disease progression.

The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB2 subtype of cannabinoid receptor during non-immune-mediated pathological conditions, little is known about the expression of the cannabinoid system during immune-mediated CNS pathology. To examine this question, we measured CB2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real-time PCR, found a 100-fold increase in CB2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB2 receptor during EAE, and found that activated microglial cells expressed 10-fold more CB2 receptor than microglia in the resting state. To determine the signals required for the up-regulation of the CB2 receptor, we cultured microglial cells with combinations of gamma-interferon (IFN-gamma) and granulocyte) macrophage-colony stimulating factor (GM-CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10-fold increase in the CB2 receptor. We found no difference in the amount of the CB2 receptor ligand, 2-arachidonylglycerol (2-AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB2 receptor up-regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune-induced inflammation.

Microglial cells are central nervous system (CNS) resident cells that are thought to become activated and contribute to the inflammation that occurs in the human autoimmune disease multiple sclerosis (MS). This has never been proven, however, because microglial cells cannot be phenotypically distinguished from peripheral macrophages that accumulate in MS inflammatory lesions. To study the kinetics and nature of microglial cell activation in the CNS, we used the animal model of MS, experimental autoimmune encephalomyelitis (EAE), and induced EAE in bone marrow (BM) chimera mice generated using major histocompatibility complex (MHC)-mismatched donor BM, allowing the separation of microglial cells and peripheral monocytes/macrophages. We found that microglial cell activation was evident before onset of disease symptoms and infiltration of peripheral myeloid cells into the CNS. Activated microglial cells underwent proliferation and upregulated the expression of CD45, MHC class II, CD40, CD86, and the dendritic cell marker CD11c. At the peak of EAE disease, activated microglial cells comprised 37% of the total macrophage and dendritic cell populations and colocalized with infiltrating leukocytes in inflammatory lesions. Our findings thus definitively demonstrate that during EAE, microglial cells become activated early in EAE disease and then differentiate into both macrophages and dendritic-like cells, suggesting they play an active role in the pathogenesis of EAE and MS., ((c) 2005 Wiley-Liss, Inc.)

Microglial cells constitute what is considered to be a fixed macrophage population in the central nervous system (CNS), which are broadly implicated in the regulation of neuroinflammation. In the normal adult CNS, microglial cells exist in a resting state characterized by a minimal or negative expression of MHC class II and the co-stimulatory molecules CD80, CD86 and CD40 and exhibit a unique ramified morphology. Microglial cell activation is associated with many inflammatory and neurogenerative CNS pathologies and is characterized by the transformation of resting microglia into cells with a macrophage morphology and up-regulation of MHC class II and co-stimulatory molecules. The cellular and molecular mechanisms required for microglial cell activation and their immunological functions in the adult brain still remain enigmatic, primarily due to the lack of an appropriate culture system that both facilitates microglial survival and expansion in the resting state. Here, we describe a new M-CSF-dependent culture system that overcomes these barriers and allows the long-term proliferation and maintenance of resting adult microglial cells isolated from the CNS. These cultured microglial cells retain their plasticity as indicated by their ability to up-regulate MHC class II and differentiate into cells with a macrophage morphology following the addition of IFN-gamma and GM-CSF, or activated T cells, which produce both cytokines. By measuring the proliferation of the T cells, we were also able to demonstrate that the microglial cells differentiated into fully functional antigen presenting cells. In addition, the replacement of the M-CSF with GM-CSF resulted in the differentiation of microglial cells into cells morphologically and phenotypically similar to dendritic cells. Our microglial cell culture system is the first described that allows the expansion of adult cells in the resting state and will facilitate studies examining the specific mechanisms of microglial cell activation and functions involved in a variety of CNS pathologies.

Gamma delta T cells have been shown to regulate immune responses associated with inflammation, but the mechanism of this regulation is largely unknown. Using the experimental autoimmune encephalomyelitis (EAE) model of the human CNS autoimmune disease multiple sclerosis, we demonstrate that gamma delta T cells are important regulators of CNS inflammation. This was shown using gamma delta T cell-deficient mice that were unable to recover from EAE. The chronic disease was accompanied by a prolonged presence of both macrophages and lymphocytes in the CNS. This extended inflammatory response was due to alterations in both cell proliferation and death. In mice lacking gamma delta T cells, proliferation of encephalitogenic T cells was 3-fold higher, and caspase activity, indicating apoptosis, was 2-fold lower compared with those in control mice recovering from EAE. gamma delta T cell-deficient mice reconstituted with wild-type gamma delta T cells recovered from EAE and resolved inflammation in the CNS, whereas mice reconstituted with Fas ligand-dysfunctional gamma delta T cells did not. Thus, gamma delta T cells regulate both inflammation in the CNS and disease recovery via Fas/Fas ligand-induced apoptosis of encephalitogenic T cells, and a quick resolution of inflammation in the CNS is essential to prevent permanent damage to the CNS resulting in chronic disease.

Interferon-gamma has been shown to be important for the resolution of inflammation associated with CNS autoimmunity. Because one of the roles of gamma delta T cells is the regulation of inflammation, we asked whether gamma delta T cells were able to regulate CNS inflammation using the autoimmune disease mouse model experimental autoimmune encephalomyelitis (EAE). We show that the presence of gamma delta T cells was needed to promote the production of IFN-gamma by both CD4 and CD8 T cells in the CNS before the onset of EAE. This regulation was shown to be independent of the ability of gamma delta T cells to produce IFN-gamma, and was specific to T cells in the CNS, as no alterations in IFN-gamma production were detectable in gamma delta T cell-deficient mice in the spleen and lymph nodes of mice with EAE or following immunization. Analysis of TCR gamma delta gene usage in the CNS showed that the only TCR delta V gene families present in the CNS before EAE onset are from the DV7s6 and DV105s1 gene families. We also show that the primary IFN-gamma-producing cells in the CNS are the encephalitogenic T cells, and that gamma delta T cell-deficient mice are unable to resolve EAE disease symptoms like control mice, thus exhibiting a long-term chronic disease course similar to that observed in IFN-gamma-deficient mice. These data suggest that CNS resident gamma delta T cells promote the production of IFN-gamma by encephalitogenic T cells in the CNS, which is ultimately required for the recovery from EAE.

Heat shock proteins (HSPs) are involved in a variety of intracellular processes and can have both pro- and anti-apoptotic action. However, little is known about the role of HSPs in the progression of apoptosis. Translocation of HSPs to the surface of apoptotic cells is a previously observed phenomenon demonstrating participation of these proteins in execution of the terminal stages of apoptosis. In a previous study we showed that development of EL-4 lymphoma cell apoptosis in vitro is accompanied by elevation of surface HSP expression. In this study we used this model to analyse the relationship of HSP70 expression and its translocation to the cell surface during apoptosis with some key intracellular events. Our data demonstrate a synchronization of surface and intracellular HSP70 expression with bcl-2 expression, intracellular reactive oxygen species (ROS) concentration and caspase-3 activity. A maximum level of surface and intracellular HSP70 expression was observed at an irreversible phase of EL-4 cell apoptosis after mitochondrial potential depolarization. In addition, an enhancement of the relative level of cytoplasmic HSP70 translocation to the cell surface was concomitant with EL-4 cell apoptosis. However, the size of surface and intracellular pools of HSP70, increasing for initial and intermediate stages of cell death, decreased at the terminal phase of apoptosis. Western blot analysis of HSP70 in conditioned supernatant obtained from EL-4 cell tissue showed that the observed decrease of HSP70 cell content might be due to surface HSP70 shedding into the intercellular space.