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Abstract Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer’s Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~50% of the mass of diffusible Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aβ tune disease-related functions of Aβ aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Constituents of air pollution, the ultrafine particles (UFP) with a diameter of ≤0.1 μm, are considerably related to traffic emissions. Several studies link air pollution to Alzheimer's disease (AD), yet the exact relationship between the two remains poorly understood. Mitochondria are known targets of environmental toxicants, and their dysfunction is associated with neurodegenerative diseases. The olfactory mucosa (OM), located at the rooftop of the nasal cavity, is directly exposed to the environment and in contact with the brain. Mounting evidence suggests that the UFPs can impact the brain directly through the olfactory tract. By using primary human OM cultures established from nasal biopsies of cognitively healthy controls and individuals diagnosed with AD, we aimed to decipher the effects of traffic-related UFPs on mitochondria. The UFP samples were collected from the exhausts of a modern heavy-duty diesel engine (HDE) without aftertreatment systems, run with renewable diesel (A0) and petroleum diesel (A20), and from an engine of a 2019 model diesel passenger car (DI-E6d) equipped with state-of-the-art aftertreatment devices and run with renewable diesel (Euro6). OM cells were exposed to three different UFPs for 24-h and 72-h, after which cellular processes were assessed on the functional and transcriptomic levels. Our results show that UFPs impair mitochondrial functions in primary human OM cells by hampering oxidative phosphorylation (OXPHOS) and redox balance, and the responses of AD cells differ from cognitively healthy controls. RNA-Seq and IPA® revealed inhibition of OXPHOS and mitochondrial dysfunction in response to UFPs A0 and A20. Functional validation confirmed that A0 and A20 impair cellular respiration, decrease ATP levels, and disturb redox balance by altering NAD and glutathione metabolism, leading to increased ROS and oxidative stress. RNA-Seq and functional assessment revealed the presence of AD-related alterations in human OM cells and that different fuels and engine technologies elicit differential effects.

Abstract Background Air pollution is recognized as an emerging environmental risk factor for neurological diseases. Large-scale epidemiological studies associate traffic-related particulate matter (PM) with impaired cognitive functions and increased incidence of neurodegenerative diseases such as Alzheimer’s disease. Inhaled components of PM may directly invade the brain via the olfactory route, or act through peripheral system responses resulting in inflammation and oxidative stress in the brain. Microglia are the immune cells of the brain implicated in the progression of neurodegenerative diseases. However, it remains unknown how PM affects live human microglia. Results Here we show that two different PMs derived from exhausts of cars running on EN590 diesel or compressed natural gas (CNG) alter the function of human microglia-like cells in vitro. We exposed human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGLs) to traffic related PMs and explored their functional responses. Lower concentrations of PMs ranging between 10 and 100 µg ml−1 increased microglial survival whereas higher concentrations became toxic over time. Both tested pollutants impaired microglial phagocytosis and increased secretion of a few proinflammatory cytokines with distinct patterns, compared to lipopolysaccharide induced responses. iMGLs showed pollutant dependent responses to production of reactive oxygen species (ROS) with CNG inducing and EN590 reducing ROS production. Conclusions Our study indicates that traffic-related air pollutants alter the function of human microglia and warrant further studies to determine whether these changes contribute to adverse effects in the brain and on cognition over time. This study demonstrates human iPSC-microglia as a valuable tool to study functional microglial responses to environmental agents.

The Patient Similarity Network paradigm implies modeling the similarity between patients based on specific data. The similarity can summarize patients' relationships from high-dimensional data, such as biological omics. The end PSN can undergo un/supervised learning tasks while being strongly interpretable, tailored for precision medicine, and ready to be analyzed with graph-theory methods. However, these benefits are not guaranteed and depend on the granularity of the summarized data, the clarity of the similarity measure, the complexity of the network's topology, and the implemented methods for analysis. To date, no patient classifier fully leverages the paradigm's inherent benefits. PSNs remain complex, unexploited, and meaningless. We present StellarPath, a hierarchical-vertical patient classifier that leverages pathway analysis and patient similarity concepts to find meaningful features for both classes and individuals. StellarPath processes omics data, hierarchically integrates them into pathways, and uses a novel similarity to measure how patients' pathway activity is alike. It selects biologically relevant molecules, pathways, and networks, considering molecule stability and topology. A graph convolutional neural network then predicts unknown patients based on known cases. StellarPath excels in classification performances and computational resources across sixteen datasets. It demonstrates proficiency in inferring the class of new patients described in external independent studies, following its initial training and testing phases on a local dataset. It advances the PSN paradigm and provides new markers, insights, and tools for in-depth patient profiling.

Abstract LRRK2-G2019S is one of the most common Parkinson’s disease (PD)-associated mutations and has been shown to alter microglial functionality. However, the impact of LRRK2-G2019S on transcriptional profile of human induced pluripotent stem cell-derived microglia-like cells (iMGLs) and how it corresponds to microglia in idiopathic PD brain is not known. Here we demonstrate that LRRK2-G2019S carrying iMGL recapitulate aspects of the transcriptional signature of human idiopathic PD midbrain microglia. LRRK2-G2019S induced subtle and donor-dependent alterations in iMGL mitochondrial respiration, phagocytosis and cytokine secretion. Investigation of microglial transcriptional state in the midbrains of PD patients revealed a subset of microglia with a transcriptional overlap between the in vitro PD-iMGL and human midbrain PD microglia. We conclude that LRRK2-G2019S iMGL serve as a model to study PD-related effects in human microglia.

Summary: Brain functionality relies on finely tuned regulation of gene expression by networks of non-coding RNAs (ncRNAs) such as the one composed by the circular RNA ciRS-7 (also known as CDR1as), the microRNA miR-7, and the long ncRNA Cyrano. We describe ischemia-induced alterations in the ncRNA network both in vitro and in vivo and in transgenic mice lacking ciRS-7 or miR-7. Our data show that cortical neurons downregulate ciRS-7 and Cyrano and upregulate miR-7 expression during ischemia. Mice lacking ciRS-7 exhibit reduced lesion size and motor impairment, while the absence of miR-7 alone results in increased ischemia-induced neuronal death. Moreover, miR-7 levels in pyramidal excitatory neurons regulate neurite morphology and glutamatergic signaling, suggesting a potential molecular link to the in vivo phenotype. Our data reveal the role of ciRS-7 and miR-7 in modulating ischemic stroke outcome, shedding light on the pathophysiological function of intracellular ncRNA networks in the brain.

Recent studies have shown that obesity and insulin resistance are associated with increased insulin-stimulated glucose uptake (GU) in the brain. Thus, insulin sensitivity seems to work differently in the brain compared to the peripheral tissues like skeletal muscles, but the underlying mechanisms remain unknown. Regular exercise training improves skeletal muscle and whole-body insulin sensitivity. However, the effect of exercise on glucose metabolism in the brain and internal organs is less well understood. The CROSRAT study aims to investigate the effects of exercise training on brain glucose metabolism and inflammation in a high-fat diet-induced rat model of obesity and insulin resistance. Male Sprague Dawley rats (n = 144) are divided into nine study groups that undergo different dietary and/or exercise training interventions lasting 12 to 24 weeks. Insulin-stimulated GU from various tissues and brain inflammation are investigated using [18F]FDG-PET/CT and [11C]PK11195-PET/CT, respectively. In addition, peripheral tissue, brain, and fecal samples are collected to study the underlying mechanisms. The strength of this study design is that it allows examining the effects of both diet and exercise training on obesity-induced insulin resistance and inflammation. As the pathophysiological changes are studied simultaneously in many tissues and organs at several time points, the study provides insight into when and where these pathophysiological changes occur.

Abstract Background Microglia are the endogenous immune cells of the brain and act as sensors of pathology to maintain brain homeostasis and eliminate potential threats. In Alzheimer's disease (AD), toxic amyloid beta (Aβ) accumulates in the brain and forms stiff plaques. In late-onset AD accounting for 95% of all cases, this is thought to be due to reduced clearance of Aβ. Human genome-wide association studies and animal models suggest that reduced clearance results from aberrant function of microglia. While the impact of neurochemical pathways on microglia had been broadly studied, mechanical receptors regulating microglial functions remain largely unexplored. Methods Here we showed that a mechanotransduction ion channel, PIEZO1, is expressed and functional in human and mouse microglia. We used a small molecule agonist, Yoda1, to study how activation of PIEZO1 affects AD-related functions in human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGL) under controlled laboratory experiments. Cell survival, metabolism, phagocytosis and lysosomal activity were assessed using real-time functional assays. To evaluate the effect of activation of PIEZO1 in vivo, 5-month-old 5xFAD male mice were infused daily with Yoda1 for two weeks through intracranial cannulas. Microglial Iba1 expression and Aβ pathology were quantified with immunohistochemistry and confocal microscopy. Published human and mouse AD datasets were used for in-depth analysis of PIEZO1 gene expression and related pathways in microglial subpopulations. Results We show that PIEZO1 orchestrates Aβ clearance by enhancing microglial survival, phagocytosis, and lysosomal activity. Aβ inhibited PIEZO1-mediated calcium transients, whereas activation of PIEZO1 with a selective agonist, Yoda1, improved microglial phagocytosis resulting in Aβ clearance both in human and mouse models of AD. Moreover, PIEZO1 expression was associated with a unique microglial transcriptional phenotype in AD as indicated by assessment of cellular metabolism, and human and mouse single-cell datasets. Conclusion These results indicate that the compromised function of microglia in AD could be improved by controlled activation of PIEZO1 channels resulting in alleviated Aβ burden. Pharmacological regulation of these mechanoreceptors in microglia could represent a novel therapeutic paradigm for AD.

Abstract A system of lymphatic vessels has been recently characterized in the meninges, with a postulated role in ‘cleaning’ the brain via cerebral fluid drainage. As meninges are the origin site of migraine pain, we hypothesized that malfunctioning of the lymphatic system should affect the local trigeminal nociception. To test this hypothesis, we studied nociceptive and inflammatory mechanisms in the hemiskull preparations (containing the meninges) of K14-VEGFR3-Ig (K14) mice lacking the meningeal lymphatic system. We recorded the spiking activity of meningeal afferents and estimated the local mast cells population, calcitonin gene-related peptide (CGRP) and cytokine levels as well as the dural trigeminal innervation in freshly-isolated hemiskull preparations from K14-VEGFR3-Ig (K14) or wild type C57BL/6 mice (WT). Spiking activity data have been confirmed in an acquired model of meningeal lymphatic dysfunction (AAV-mVEGFR3(1–4)Ig induced lymphatic ablation). We found that levels of the pro-inflammatory cytokine IL12-p70 and CGRP, implicated in migraine, were reduced in the meninges of K14 mice, while the levels of the mast cell activator MCP-1 were increased. The other migraine-related pro-inflammatory cytokines (basal and stimulated), did not differ between the two genotypes. The patterns of trigeminal innervation in meninges remained unchanged and we did not observe alterations in basal or ATP-induced nociceptive firing in the meningeal afferents associated with meningeal lymphatic dysfunction. In summary, the lack of meningeal lymphatic system is associated with a new balance between pro- and anti-migraine mediators but does not directly trigger meningeal nociceptive state.

Abstract Hypoxia induces changes in the secretion of extracellular vesicles (EVs) in several non‐neuronal cells and pathological conditions. EVs are packed with biomolecules, such as microRNA(miR)‐21‐5p, which respond to hypoxia. However, the true EV association of miR‐21‐5p, and its functional or biomarker relevance, are inadequately characterised. Neurons are extremely sensitive cells, and it is not known whether the secretion of neuronal EVs and miR‐21‐5p are altered upon hypoxia. Here, we characterised the temporal EV secretion profile and cell viability of neurons under hypoxia. Hypoxia induced a rapid increase of miR‐21a‐5p secretion in the EVs, which preceded the elevation of hypoxia‐induced tissue or cellular miR‐21a‐5p. Prolonged hypoxia induced cell death and the release of morphologically distinct EVs. The EVs protected miR‐21a‐5p from enzymatic degradation but a remarkable fraction of miR‐21a‐5p remained fragile and non‐EV associated. The increase in miR‐21a‐5p secretion may have biomarker potential, as high blood levels of miR‐21‐5p in stroke patients were associated with significant disability at hospital discharge. Our data provides an understanding of the dynamic regulation of EV secretion from neurons under hypoxia and provides a candidate for the prediction of recovery from ischemic stroke.

Under physiological conditions in vivo astrocytes internalize and degrade neuronal mitochondria in a process called transmitophagy. Mitophagy is widely reported to be impaired in neurodegeneration but it is unknown whether and how transmitophagy is altered in Alzheimer's disease (AD). Here we report that the internalization of neuronal mitochondria is significantly increased in astrocytes isolated from AD mouse brains. We also demonstrate that the degradation of neuronal mitochondria by astrocytes is increased in AD mice at the age of 6 months onwards. Furthermore, we demonstrate for the first time a similar phenomenon between human neurons and AD astrocytes, and in murine hippocampi in vivo. The results suggest the involvement of S100a4 in impaired mitochondrial transfer between neurons and AD astrocytes together with significant increases in the mitophagy regulator and reactive oxygen species in aged AD astrocytes. These findings demonstrate altered neuron-supporting functions of AD astrocytes and provide a starting point for studying the molecular mechanisms of transmitophagy in AD.

Abstract Peripheral infections followed by systemic inflammation may contribute to the onset of Alzheimer`s disease (AD) and accelerate the disease progression later in life. Yet, the impact of systemic inflammation on the plasma and brain tissue metabolome and lipidome in AD has not been investigated. In this study, targeted metabolomic and untargeted lipidomic profiling experiments were performed on the plasma, cortices, and hippocampi of wild-type (WT) mice and transgenic APdE9 mice after chronic lipopolysaccharide (LPS) treatment, as well as saline-treated APdE9 mice. The lipidome and the metabolome of these mice were compared to saline-treated WT animals. In the brain tissue of all three models, the lipidome was more influenced than the metabolome. The LPS-treated APdE9 mice had the highest number of changes in brain metabolic pathways with significant alterations in levels of lysine, myo-inositol, spermine, phosphocreatine, acylcarnitines and diacylglycerols, which were not observed in the saline-treated APdE9 mice. In the WT mice, the effect of the LPS administration on metabolome and lipidome was negligible. The study provided exciting information about the biochemical perturbations due to LPS-induced inflammation in the transgenic AD model, which can significantly enhance our understanding of the role of systemic inflammation in AD pathogenesis.

Changes in brain prolyl oligopeptidase (PREP) expression and activity have been associated with neuroinflammation and Alzheimer`s disease (AD). The role of PREP in AD, which onset can be contributed by peripheral infection-induced inflammation, is unknown. The aim of the study was to elucidate further the association of PREP with AD pathology and inflammation. Here, we quantitated PREP protein expression by liquid chromatography-tandem mass spectrometry-based quantitative targeted absolute proteomics and determined PREP peptidase activity in the cerebral cortices of familial AD (APdE9) and lipopolysaccharide (LPS)-induced inflammation mouse models. PREP activity was investigated using the fluorogenic substrate Suc-Gly-Pro-AMC. PREP expression was significantly increased (by 2-fold) in the brain cytosolic fraction of LPS treated APdE9 mice, while the peptidase activity remained unaltered. In the cortical crude membrane fraction, the PREP expression and activity were decreased by 35–40% in the LPS treated APdE9 mice. In conclusion, cortical cytosolic and membrane-bound PREP expression levels and enzyme activities were altered due to LPS-induced inflammation in the AD mouse model. Since the cytosolic protein expression increased without any concomitant increase in the peptidase activity, it is likely that PREP activity is affected by other factors than protein expression alone.

Abstract Background Obesity and physical inactivity are major global public health concerns, both of which increase the risk of insulin resistance and type 2 diabetes. Regulation of glucose homeostasis involves cross-talk between the central nervous system, peripheral tissues, and gut microbiota, and is affected by genetics. Systemic cross-talk between brain, gut, and peripheral tissues in glucose homeostasis: effects of exercise training (CROSSYS) aims to gain new systems-level understanding of the central metabolism in human body, and how exercise training affects this cross-talk. Methods CROSSYS is an exercise training intervention, in which participants are monozygotic twins from pairs discordant for body mass index (BMI) and within a pair at least the other is overweight. Twins are recruited from three population-based longitudinal Finnish twin studies, including twins born in 1983–1987, 1975–1979, and 1945–1958. The participants undergo 6-month-long exercise intervention period, exercising four times a week (including endurance, strength, and high-intensity training). Before and after the exercise intervention, comprehensive measurements are performed in Turku PET Centre, Turku, Finland. The measurements include: two positron emission tomography studies (insulin-stimulated whole-body and tissue-specific glucose uptake and neuroinflammation), magnetic resonance imaging (brain morphology and function, quantification of body fat masses and organ volumes), magnetic resonance spectroscopy (quantification of fat within heart, pancreas, liver and tibialis anterior muscle), echocardiography, skeletal muscle and adipose tissue biopsies, a neuropsychological test battery as well as biosamples from blood, urine and stool. The participants also perform a maximal exercise capacity test and tests of muscular strength. Discussion This study addresses the major public health problems related to modern lifestyle, obesity, and physical inactivity. An eminent strength of this project is the possibility to study monozygotic twin pairs that share the genome at the sequence level but are discordant for BMI that is a risk factor for metabolic impairments such as insulin resistance. Thus, this exercise training intervention elucidates the effects of obesity on metabolism and whether regular exercise training is able to reverse obesity-related impairments in metabolism in the absence of the confounding effects of genetic factors. Trial registration ClinicalTrials.gov , NCT03730610 . Prospectively registered 5 November 2018.

Abstract Lipid peroxidation-initiated ferroptosis is an iron-dependent mechanism of programmed cell death taking place in neurological diseases. Here we show that a condensed benzo[b]thiazine derivative small molecule with an arylthiazine backbone (ADA-409-052) inhibits tert-Butyl hydroperoxide (TBHP)-induced lipid peroxidation (LP) and protects against ferroptotic cell death triggered by glutathione (GSH) depletion or glutathione peroxidase 4 (GPx4) inhibition in neuronal cell lines. In addition, ADA-409-052 suppresses pro-inflammatory activation of BV2 microglia and protects N2a neuronal cells from cell death induced by pro-inflammatory RAW 264.7 macrophages. Moreover, ADA-409-052 efficiently reduces infarct volume, edema and expression of pro-inflammatory genes in a mouse model of thromboembolic stroke. Targeting ferroptosis may be a promising therapeutic strategy in neurological diseases involving severe neuronal death and neuroinflammation.

Extracellular vesicles (EVs) naturally carry cargo from producer cells, such as RNA and protein, and can transfer these messengers to other cells and tissue. This ability provides an interesting opportunity for using EVs as delivery vehicles for therapeutic agents, such as for gene therapy. However, endogenous loading of cargo, such as microRNAs (miRNAs), is not very efficient as the copy number of miRNAs per EV is quite low. Therefore, new methods and tools to enhance the loading of small RNAs is required. In the current study, we developed fusion protein of EV membrane protein CD9 and RNA-binding protein AGO2 (hCD9.hAGO2). We show that the EVs engineered with hCD9.hAGO2 contain significantly higher levels of miRNA or shRNA (miR-466c or shRNA-451, respectively) compared to EVs that are isolated from cells that only overexpress the desired miRNA or shRNA. These hCD9.hAGO2 engineered EVs also transfer their RNA cargo to recipient cells more efficiently. We were not able to detect changes in gene expression levels in recipient cells after the EV treatments, but we show that the cell viability of HUVECs was increased after hCD9.hAGO2 EV treatments. This technical study characterizes the hCD9.hAGO2 fusion protein for the future development of enhanced RNA loading to EVs.

MicroRNAs are well characterized in their role in silencing gene expression by targeting 3´-UTR of mRNAs in cytoplasm. However, recent studies have shown that miRNAs have a role in the regulation of genes in the nucleus, where they are abundantly located. We show here that in mouse endothelial cell line (C166), nuclear microRNA miR-466c participates in the regulation of vascular endothelial growth factor a (Vegfa) gene expression in hypoxia. Upregulation of Vegfa expression in response to hypoxia was significantly compromised after removal of miR-466c with CRISPR-Cas9 genomic deletion. We identified a promoter-associated long non-coding RNA on mouse Vegfa promoter and show that miR-466c directly binds to this transcript to modulate Vegfa expression. Collectively, these observations suggest that miR-466c regulates Vegfa gene transcription in the nucleus by targeting the promoter, and expands on our understanding of the role of miRNAs well beyond their canonical role.

Abstract Background Increased physical exercise improves cognitive function and reduces pathology associated with Alzheimer’s disease (AD). However, the mechanisms underlying the beneficial effects of exercise in AD on the level of specific brain cell types remain poorly investigated. The involvement of astrocytes in AD pathology is widely described, but their exact role in exercise-mediated neuroprotection warrant further investigation. Here, we investigated the effect of long-term voluntary physical exercise on the modulation of the astrocyte state. Methods Male 5xFAD mice and their wild-type littermates had free access to a running wheel from 1.5 to 7 months of age. A battery of behavioral tests was used to assess the effects of voluntary exercise on cognition and learning. Neuronal loss, impairment in neurogenesis, beta-amyloid (Aβ) deposition, and inflammation were evaluated using a variety of histological and biochemical measurements. Sophisticated morphological analyses were performed to delineate the specific involvement of astrocytes in exercise-induced neuroprotection in the 5xFAD mice. Results Long-term voluntary physical exercise reversed cognitive impairment in 7-month-old 5xFAD mice without affecting neurogenesis, neuronal loss, Aβ plaque deposition, or microglia activation. Exercise increased glial fibrillary acid protein (GFAP) immunoreactivity and the number of GFAP-positive astrocytes in 5xFAD hippocampi. GFAP-positive astrocytes in hippocampi of the exercised 5xFAD mice displayed increases in the numbers of primary branches and in the soma area. In general, astrocytes distant from Aβ plaques were smaller in size and possessed simplified processes in comparison to plaque-associated GFAP-positive astrocytes. Morphological alterations of GFAP-positive astrocytes occurred concomitantly with increased astrocytic brain-derived neurotrophic factor (BDNF) and restoration of postsynaptic protein PSD-95. Conclusions Voluntary physical exercise modulates the reactive astrocyte state, which could be linked via astrocytic BDNF and PSD-95 to improved cognition in 5xFAD hippocampi. The molecular pathways involved in this modulation could potentially be targeted for benefit against AD.

Abstract Background Ischemic stroke is a devastating disease without a cure. The available treatments for ischemic stroke, thrombolysis by tissue plasminogen activator, and thrombectomy are suitable only to a fraction of patients and thus novel therapeutic approaches are urgently needed. The neuroinflammatory responses elicited secondary to the ischemic attack further aggravate the stroke-induced neuronal damage. It has been demonstrated that these responses are regulated at the level of non-coding RNAs, especially miRNAs. Methods We utilized lentiviral vectors to overexpress miR-669c in BV2 microglial cells in order to modulate their polarization. To detect whether the modulation of microglial activation by miR-669c provides protection in a mouse model of transient focal ischemic stroke, miR-669c overexpression was driven by a lentiviral vector injected into the striatum prior to induction of ischemic stroke. Results Here, we demonstrate that miR-669c-3p, a member of chromosome 2 miRNA cluster (C2MC), is induced upon hypoxic and excitotoxic conditions in vitro and in two different in vivo models of stroke. Rather than directly regulating the neuronal survival in vitro, miR-669c is capable of attenuating the microglial proinflammatory activation in vitro and inducing the expression of microglial alternative activation markers arginase 1 (Arg1), chitinase-like 3 (Ym1), and peroxisome proliferator-activated receptor gamma (PPAR-γ). Intracerebral overexpression of miR-669c significantly decreased the ischemia-induced cell death and ameliorated the stroke-induced neurological deficits both at 1 and 3 days post injury (dpi). Albeit miR-669c overexpression failed to alter the overall Iba1 protein immunoreactivity, it significantly elevated Arg1 levels in the ischemic brain and increased colocalization of Arg1 and Iba1. Moreover, miR-669c overexpression under cerebral ischemia influenced several morphological characteristics of Iba1 positive cells. We further demonstrate the myeloid differentiation primary response gene 88 (MyD88) transcript as a direct target for miR-669c-3p in vitro and show reduced levels of MyD88 in miR-669c overexpressing ischemic brains in vivo. Conclusions Collectively, our data provide the evidence that miR-669c-3p is protective in a mouse model of ischemic stroke through enhancement of the alternative microglial/macrophage activation and inhibition of MyD88 signaling. Our results accentuate the importance of controlling miRNA-regulated responses for the therapeutic benefit in conditions of stroke and neuroinflammation.

Abstract Background The adverse effects of air pollutants including particulate matter (PM) on the central nervous system is increasingly reported by epidemiological, animal and post-mortem studies in the last decade. Oxidative stress and inflammation are key consequences of exposure to PM although little is known of the exact mechanism. The association of PM exposure with deteriorating brain health is speculated to be driven by PM entry via the olfactory system. How air pollutants affect this key entry site remains elusive. In this study, we investigated effects of urban size-segregated PM on a novel cellular model: primary human olfactory mucosal (hOM) cells. Results Metabolic activity was reduced following 24-h exposure to PM without evident signs of toxicity. Results from cytometric bead array suggested a mild inflammatory response to PM exposure. We observed increased oxidative stress and caspase-3/7 activity as well as perturbed mitochondrial membrane potential in PM-exposed cells. Mitochondrial dysfunction was further verified by a decrease in mitochondria-dependent respiration. Transient suppression of the mitochondria-targeted gene, neuronal pentraxin 1 (NPTX1), was carried out, after being identified to be up-regulated in PM2.5–1 treated cells via RNA sequencing. Suppression of NPTX1 in cells exposed to PM did not restore mitochondrial defects resulting from PM exposure. In contrast, PM-induced adverse effects were magnified in the absence of NPTX1, indicating a critical role of this protein in protection against PM effects in hOM cells. Conclusion Key mitochondrial functions were perturbed by urban PM exposure in a physiologically relevant cellular model via a mechanism involving NPTX1. In addition, inflammatory response and early signs of apoptosis accompanied mitochondrial dysfunction during exposure to PM. Findings from this study contribute to increased understanding of harmful PM effects on human health and may provide information to support mitigation strategies targeted at air pollution.

The PSEN1 ΔE9 mutation causes a familial form of Alzheimer’s disease (AD) by shifting the processing of amyloid precursor protein (APP) towards the generation of highly amyloidogenic Aβ42 peptide. We have previously shown that the PSEN1 ΔE9 mutation in human-induced pluripotent stem cell (iPSC)-derived astrocytes increases Aβ42 production and impairs cellular responses. Here, we injected PSEN1 ΔE9 mutant astrosphere-derived glial progenitors into newborn mice and investigated mouse behavior at the ages of 8, 12, and 16 months. While we did not find significant behavioral changes in younger mice, spatial learning and memory were paradoxically improved in 16-month-old PSEN1 ΔE9 glia-transplanted male mice as compared to age-matched isogenic control-transplanted animals. Memory improvement was associated with lower levels of soluble, but not insoluble, human Aβ42 in the mouse brain. We also found a decreased engraftment of PSEN1 ΔE9 mutant cells in the cingulate cortex and significant transcriptional changes in both human and mouse genes in the hippocampus, including the extracellular matrix-related genes. Overall, the presence of PSEN1 ΔE9 mutant glia exerted a more beneficial effect on aged mouse brain than the isogenic control human cells likely as a combination of several factors.

Cytosolic phospholipase A2 (cPLA2) is an enzyme regulating membrane phospholipid homeostasis and the release of arachidonic acid utilized in inflammatory responses. It represents an attractive target for the treatment of Alzheimer’s disease (AD). Previously, we showed that lipopolysaccharide (LPS)-induced systemic inflammation caused abnormal lipid metabolism in the brain of a transgenic AD mouse model (APdE9), which might be associated with potential changes in cPLA2 activity. Here, we investigated changes in cPLA2 expression and activity, as well as the molecular mechanisms underlying these alterations due to chronic LPS administration in the cerebral cortex of female APdE9 mice as compared to saline- and LPS-treated female wild-type mice and saline-treated APdE9 mice. The study revealed the significant effects of genotype LPS treatment on cortical cPLA2 protein expression and activity in APdE9 mice. LPS treatment resulted in nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activation in the cortex of APdE9 mice. The gene expressions of inflammation markers Il1b and Tnfa were significantly elevated in the cortex of both APdE9 groups compared to the wild-type groups. The study provides evidence of the elevated expression and activity of cPLA2 in the brain cortex of APdE9 mice after chronic LPS treatment, which could be associated with NFkB activation.

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with neuron–glia dysfunction and dysregulated miRNAs. We previously reported upregulated miR-124/miR-21 in AD neurons and their exosomes. However, their glial distribution, phenotypic alterations and exosomal spread are scarcely documented. Here, we show glial cell activation and miR-21 overexpression in mouse organotypic hippocampal slices transplanted with SH-SY5Y cells expressing the human APP695 Swedish mutation. The upregulation of miR-21 only in the CSF from a small series of mild cognitive impairment (MCI) AD patients, but not in non-AD MCI individuals, supports its discriminatory potential. Microglia, neurons, and astrocytes differentiated from the same induced pluripotent stem cells from PSEN1ΔE9 AD patients all showed miR-21 elevation. In AD neurons, miR-124/miR-21 overexpression was recapitulated in their exosomes. In AD microglia, the upregulation of iNOS and miR-21/miR-146a supports their activation. AD astrocytes manifested a restrained inflammatory profile, with high miR-21 but low miR-155 and depleted exosomal miRNAs. Their immunostimulation with C1q + IL-1α + TNF-α induced morphological alterations and increased S100B, inflammatory transcripts, sAPPβ, cytokine release and exosomal miR-21. PPARα, a target of miR-21, was found to be repressed in all models, except in neurons, likely due to concomitant miR-125b elevation. The data from these AD models highlight miR-21 as a promising biomarker and a disease-modifying target to be further explored.

Stroke can be followed by immediate severe headaches. As headaches are initiated by the activation of trigeminal meningeal afferents, we assessed changes in the activity of meningeal afferents in mice subjected to cortical photothrombosis. Cortical photothrombosis induced ipsilateral lesions of variable sizes that were associated with contralateral sensorimotor impairment. Nociceptive firing of mechanosensitive Piezo1 channels, activated by the agonist Yoda1, was increased in meningeal afferents in the ischemic hemispheres. These meningeal afferents also had a higher maximal spike frequency at baseline and during activation of the mechanosensitive Piezo1 channel by Yoda1. Moreover, in these meningeal afferents, nociceptive firing was active during the entire induction of transient receptor potential vanilloid 1 (TRPV1) channels by capsaicin. No such activation was observed on the contralateral hemi-skulls of the same group of mice or in control mice. Our data suggest the involvement of mechanosensitive Piezo1 channels capable of maintaining high-frequency spiking activity and of nociceptive TRPV1 channels in trigeminal headache pain responses after experimental ischemic stroke in mice.

Background: Brain amyloidosis does not invariably predict dementia. We hypothesized that high soluble 42-amino acid β amyloid (Aβ42) peptide levels are associated with normal cognition and hippocampal volume despite increasing brain amyloidosis. Methods: This cross-sectional study of 598 amyloid-positive participants in the Alzheimer's Disease Neuroimaging Initiative cohort examined whether levels of soluble Aβ42 are higher in amyloid-positive normal cognition (NC) individuals compared to mild cognitive impairment (MCI) and Alzheimer's disease (AD) and whether this relationship applies to neuropsychological assessments and hippocampal volume measured within the same year. All subjects were evaluated between June 2010 and February 2019. Brain amyloid positivity was defined as positron emission tomography-based standard uptake value ratio (SUVR) ≥1.08 for [18] F-florbetaben or 1.11 for [18]F-florbetapir, with higher SUVR indicating more brain amyloidosis. Analyses were adjusted for age, sex, education, APOE4, p-tau, t-tau, and centiloids levels. Findings: Higher soluble Aβ42 levels were observed in NC (864.00 pg/ml) than in MCI (768.60 pg/ml) or AD (617.46 pg/ml), with the relationship between NC, MCI, and AD maintained across all amyloid tertiles. In adjusted analysis, there was a larger absolute effect size of soluble Aβ42 than SUVR for NC (0.82 vs. 0.40) and MCI (0.60 vs. 0.26) versus AD. Each standard deviation increase in Aβ42 was associated with greater odds of NC than AD (adjusted odds ratio, 6.26; p

Neuroinflammation has a major role in several brain disorders including Alzheimer’s disease (AD), yet at present there are no effective anti-neuroinflammatory therapeutics available. Copper(II) complexes of bis(thiosemicarbazones) (CuII(gtsm) and CuII(atsm)) have broad therapeutic actions in preclinical models of neurodegeneration, with CuII(atsm) demonstrating beneficial outcomes on neuroinflammatory markers in vitro and in vivo. These findings suggest that copper(II) complexes could be harnessed as a new approach to modulate immune function in neurodegenerative diseases. In this study, we examined the anti-neuroinflammatory action of several low-molecular-weight, charge-neutral and lipophilic copper(II) complexes. Our analysis revealed that one compound, a thiosemicarbazone–pyridylhydrazone copper(II) complex (CuL5), delivered copper into cells in vitro and increased the concentration of copper in the brain in vivo. In a primary murine microglia culture, CuL5 was shown to decrease secretion of pro-inflammatory cytokine macrophage chemoattractant protein 1 (MCP-1) and expression of tumor necrosis factor alpha (Tnf), increase expression of metallothionein (Mt1), and modulate expression of Alzheimer’s disease-associated risk genes, Trem2 and Cd33. CuL5 also improved the phagocytic function of microglia in vitro. In 5xFAD model AD mice, treatment with CuL5 led to an improved performance in a spatial working memory test, while, interestingly, increased accumulation of amyloid plaques in treated mice. These findings demonstrate that CuL5 can induce anti-neuroinflammatory effects in vitro and provide selective benefit in vivo. The outcomes provide further support for the development of copper-based compounds to modulate neuroinflammation in brain diseases.

Abstract MicroRNAs (miRNAs) are small non-coding RNAs that have well-characterized roles in cytoplasmic gene regulation, where they act by binding to mRNA transcripts and inhibiting their translation (i.e. post-transcriptional gene silencing, PTGS). However, miRNAs have also been implicated in transcriptional gene regulation and alternative splicing, events that are restricted to the cell nucleus. Here we performed nuclear-cytoplasmic fractionation in a mouse endothelial cell line and characterized the localization of miRNAs in response to hypoxia using small RNA sequencing. A highly diverse population of abundant miRNA species was detected in the nucleus, of which the majority (56%) was found to be preferentially localized in one compartment or the other. Induction of hypoxia resulted in changes in miRNA levels in both nuclear and cytoplasmic compartments, with the majority of changes being restricted to one location and not the other. Notably, the classical hypoxamiR (miR-210-3p) was highly up-regulated in the nuclear compartment after hypoxic stimulus. These findings reveal a previously unappreciated level of molecular complexity in the physiological response occurring in ischemic tissue. Furthermore, widespread differential miRNA expression in the nucleus strongly suggests that these small RNAs are likely to perform extensive nuclear regulatory functions in the general case.

Inflammation is a prominent feature of the neuropathology of amyotrophic lateral sclerosis (ALS). Emerging evidence suggests that inflammatory cascades contributing to the disease progression are not restricted to the central nervous system (CNS) but also occur peripherally. Indeed, alterations in T cell responses and their secreted cytokines have been detected in ALS patients and in animal models of ALS. One key cytokine responsible for the shift in T cell responses is interleukin-33 (IL-33), which stimulates innate type 2 immune cells to produce a large amount of Th2 cytokines that are possibly beneficial in the recovery processes of CNS injuries. Since the levels of IL-33 have been shown to be decreased in patients affected with ALS, we sought to determine whether a long-term recombinant IL-33 treatment of a transgenic mouse model of ALS expressing G93A-superoxide dismutase 1 (SOD1-G93A) alters the disease progression and ameliorates the ALS-like disease pathology. SOD1-G93A mice were treated with intraperitoneal injections of IL-33 and effects on disease onset and inflammatory status were determined. Spinal cord (SC) neurons, astrocytes and T-cells were exposed to IL-33 to evaluate the cell specific responses to IL-33. Treatment of SOD1-G93A mice with IL-33 delayed the disease onset in female mice, decreased the proportion of CD4+ and CD8 + T cell populations in the spleen and lymph nodes, and alleviated astrocytic activation in the ventral horn of the lumbar SC. Male SOD1-G93A mice were unresponsive to the treatment. In vitro studies showed that IL-33 is most likely not acting directly on neurons and astrocytes, but rather conveying its effects through peripheral T-cells. Our results suggest that strategies directed to the peripheral immune system may have therapeutic potential in ALS. The effect of gender dimorphisms to the treatment efficacy needs to be taken into consideration when designing new therapeutic strategies for CNS diseases. Keywords: ALS, Inflammation, Interleukin-33, Cytokine, Astrocyte, T cell, Microglia, Spinal cord

The protein corona around artificial nanoparticles is known to influence activity and biological fate, the formation around viruses is less well understood. Here, the authors observe the formation of protein corona on viruses and study the effects this corona has on viral infectivity and on amyloid protein assembly.

Piezo1 channels are highly mechanically-activated cation channels that can sense and transduce the mechanical stimuli into physiological signals in different tissues including skeletal muscle. In this focused review, we summarize the emerging evidence of Piezo1 channel-mediated effects in the physiology of skeletal muscle, with a particular focus on the role of Piezo1 in controlling myogenic precursor activity and skeletal muscle regeneration and vascularization. The disclosed effects reported by pharmacological activation of Piezo1 channels with the selective agonist Yoda1 indicate a potential impact of Piezo1 channel activity in skeletal muscle regeneration, which is disrupted in various muscular pathological states. All findings reported so far agree with the idea that Piezo1 channels represent a novel, powerful molecular target to develop new therapeutic strategies for preventing or ameliorating skeletal muscle disorders characterized by an impairment of tissue regenerative potential.

Glial cells are crucial for the maintenance of correct neuronal functionality in a physiological state and intervene to restore the equilibrium when environmental or pathological conditions challenge central nervous system homeostasis. The communication between glial cells and neurons is essential and extracellular vesicles (EVs) take part in this function by transporting a plethora of molecules with the capacity to influence the function of the recipient cells. EVs, including exosomes and microvesicles, are a heterogeneous group of biogenetically distinct double membrane-enclosed vesicles. Once released from the cell, these two types of vesicles are difficult to discern, thus we will call them with the general term of EVs. This review is focused on the EVs secreted by astrocytes, oligodendrocytes and microglia, aiming to shed light on their influence on neurons and on the overall homeostasis of the central nervous system functions. We collect evidence on neuroprotective and homeostatic effects of glial EVs, including neuronal plasticity. On the other hand, current knowledge of the detrimental effects of the EVs in pathological conditions is addressed. Finally, we propose directions for future studies and we evaluate the potential of EVs as a therapeutic treatment for neurological disorders.

Background: Extracellular ATP is a powerful trigger of neuroinflammation by activating immune cells via P2X7 receptors. Acetylcholine and nicotinic agonists inhibit ATP-triggered proinflammatory cytokines via the so-called “cholinergic anti-inflammatory pathway” (CAP). However, it remains unclear as to what stage of ATP-induced signaling cholinergic agents provide this anti-inflammatory effect. Using the specific property of P2X7 receptor to open a pathway permeable to large molecules, associated with activation of inflammasome, we studied the action of cholinergic agents on this key event in CAP activation.Methods: Freshly isolated mouse peritoneal mast cells and primary human macrophages were used. To assess P2X7 channel opening, the permeability to the fluorescent dye YO-PRO1 or ethidium bromide (EtBr) was measured by flow cytometry. Expression of nicotinic receptors was probed in macrophages with the fluorescently labeled α-bungarotoxin or with patch-clamp recordings.Results: ATP opened P2X7 ion channels in mast cells and macrophages permeable to YO-PRO1 or EtBr, respectively. This stimulatory effect in mast cells was inhibited by the specific P2X7 antagonist A839977 confirming that YO-PRO1 uptake was mediated via ATP-gated P2X7 ion channels. Cholinergic agents also slightly induced dye uptake to mast cells but not in macrophages, which expressed functional α7 nicotinic receptors. However, both in mast cells and in macrophages, acetylcholine and nicotine failed to inhibit the stimulatory effect of ATP on dye uptake.Conclusion: These data suggest that in immune cells, cholinergic agents do not act on P2X7 receptor-coupled large pore formation but can mediate the anti-inflammatory effect underlying CAP downstream of ATP-driven signaling.

Olfaction is orchestrated by olfactory mucosal cells located in the upper nasal cavity. Olfactory dysfunction manifests early in several neurodegenerative disorders including Alzheimer’s disease, however, disease-related alterations to the olfactory mucosal cells remain poorly described. The aim of this study was to evaluate the olfactory mucosa differences between cognitively healthy individuals and Alzheimer’s disease patients. We report increased amyloid-beta secretion in Alzheimer’s disease olfactory mucosal cells and detail cell-type-specific gene expression patterns, unveiling 240 differentially expressed disease-associated genes compared to the cognitively healthy controls, and five distinct cell populations. Overall, alterations of RNA and protein metabolism, inflammatory processes, and signal transduction were observed in multiple cell populations, suggesting their role in Alzheimer’s disease-related olfactory mucosa pathophysiology. Furthermore, the single-cell RNA-sequencing proposed alterations in gene expression of mitochondrially located genes in AD OM cells, which were verified by functional assays, demonstrating altered mitochondrial respiration and a reduction of ATP production. Our results reveal disease-related changes of olfactory mucosal cells in Alzheimer’s disease and demonstrate the utility of single-cell RNA sequencing data for investigating molecular and cellular mechanisms associated with the disease.

Amyloids are fibrillar protein aggregates associated with diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), type II diabetes and Creutzfeldt–Jakob disease. The process of amyloid polymerization involves three pathological protein transformations; from natively folded conformation to the cross-β conformation, from biophysically soluble to insoluble, and from biologically functional to non-functional. While amyloids share a similar cross-β conformation, the biophysical transformation can either take place spontaneously via a homogeneous nucleation mechanism (HON) or catalytically on an exogenous surface via a heterogeneous nucleation mechanism (HEN). Here, we postulate that the different nucleation pathways can serve as a mechanistic basis for an etiological classification of amyloidopathies, where hereditary forms generally follow the HON pathway, while sporadic forms follow seed-induced (prions) or surface-induced (including microbially induced) HEN pathways. Critically, the conformational and biophysical amyloid transformation results in loss-of-function (LOF) of the original natively folded and soluble protein. This LOF can, at least initially, be the mechanism of amyloid toxicity even before amyloid accumulation reaches toxic levels. By highlighting the important role of non-protein species in amyloid formation and LOF mechanisms of toxicity, we propose a generalized mechanistic framework that could help better understand the diverse etiology of amyloid diseases and offer new opportunities for therapeutic interventions, including replacement therapies.

Abstract Two of the key functions of arteries in the brain are (1) the well‐recognized supply of blood via the vascular lumen and (2) the emerging role for the arterial walls as routes for the elimination of interstitial fluid (ISF) and soluble metabolites, such as amyloid beta (Aβ), from the brain and retina. As the brain and retina possess no conventional lymphatic vessels, fluid drainage toward peripheral lymph nodes is mediated via transport along basement membranes in the walls of capillaries and arteries that form the intramural peri‐arterial drainage (IPAD) system. IPAD tends to fail as arteries age but the mechanisms underlying the failure are unclear. In some people this is reflected in the accumulation of Aβ plaques in the brain in Alzheimer's disease (AD) and deposition of Aβ within artery walls as cerebral amyloid angiopathy (CAA). Knowledge of the dynamics of IPAD and why it fails with age is essential for establishing diagnostic tests for the early stages of the disease and for devising therapies that promote the clearance of Aβ in the prevention and treatment of AD and CAA. This editorial is intended to introduce the rationale that has led to the establishment of the Clearance of Interstitial Fluid (ISF) and CSF (CLIC) group, within the Vascular Professional Interest Area of the Alzheimer's Association International Society to Advance Alzheimer's Research and Treatment.

Human pluripotent stem cell (hPSC)-derived neuron cultures have emerged as models of electrical activity in the human brain. Microelectrode arrays (MEAs) measure changes in the extracellular electric potential of cell cultures or tissues and enable the recording of neuronal network activity. MEAs have been applied to both human subjects and hPSC-derived brain models. Here, we review the literature on the functional characterization of hPSC-derived two- and three-dimensional brain models with MEAs and examine their network function in physiological and pathological contexts. We also summarize MEA results from the human brain and compare them to the literature on MEA recordings of hPSC-derived brain models. MEA recordings have shown network activity in two-dimensional hPSC-derived brain models that is comparable to the human brain and revealed pathology-associated changes in disease models. Three-dimensional hPSC-derived models such as brain organoids possess a more relevant microenvironment, tissue architecture and potential for modeling the network activity with more complexity than two-dimensional models. hPSC-derived brain models recapitulate many aspects of network function in the human brain and provide valid disease models, but certain advancements in differentiation methods, bioengineering and available MEA technology are needed for these approaches to reach their full potential.

Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.

Extracellular vesicles (EVs) effectively suppress neuroinflammation and induce neuroprotective effects in different disease models. However, the mechanisms by which EVs regulate the neuroinflammatory response of microglia remains largely unexplored. Here, we addressed this issue by testing the action of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on immortalized human microglial cells. We found that EVs induced a rapid increase in intracellular Ca2+ and promoted significant ATP release in microglial cells after 20 min of treatment. Boyden chamber assays revealed that EVs promoted microglial migration by 20%. Pharmacological inhibition of different subtypes of purinergic receptors demonstrated that EVs activated microglial migration preferentially through the P2X4 receptor (P2X4R) pathway. Proximity ligation and co-immunoprecipitation assays revealed that EVs promote association between milk fat globule-epidermal growth factor-factor VIII (MFG-E8) and P2X4R proteins. Furthermore, pharmacological inhibition of αVβ3/αVβ5 integrin suppressed EV-induced cell migration and formation of lipid rafts in microglia. These results demonstrate that EVs promote microglial motility through P2X4R/MFG-E8-dependent mechanisms. Our findings provide novel insights into the molecular mechanisms through which EVs target human microglia that may be exploited for the development of new therapeutic strategies targeting disease-associated neuroinflammation.

Abstract Background Ischemic stroke is one of the main causes of death and disability worldwide. It is caused by the cessation of cerebral blood flow resulting in the insufficient delivery of glucose and oxygen to the neural tissue. The inflammatory response initiated by ischemic stroke in order to restore tissue homeostasis in the acute phase of stroke contributes to delayed brain damage. Methods By using in vitro models of neuroinflammation and in vivo model of permanent middle cerebral artery occlusion, we demonstrate the neuroprotective and anti-inflammatory effects of sulfosuccinimidyl oleate sodium (SSO). Results SSO significantly reduced the lipopolysaccharide/interferon-γ-induced production of nitric oxide, interleukin-6 and tumor necrosis factor-α, and the protein levels of inflammatory enzymes including nitric oxide synthase 2, cyclooxygenase-2 (COX-2), and p38 mitogen-activated protein kinase (MAPK) in microglia, without causing cell toxicity. Although SSO failed to directly alleviate glutamate-induced excitotoxicity in murine cortical neurons, it prevented inflammation-induced neuronal death in microglia-neuron co-cultures. Importantly, oral administration of SSO in Balb/c mice subjected to permanent occlusion of the middle cerebral artery reduced microglial activation in the peri-ischemic area and attenuated brain damage. This in vivo neuroprotective effect of SSO was associated with a reduction in the COX-2 and heme oxygenase-1 immunoreactivities. Conclusions Our results suggest that SSO is an anti-inflammatory and a possible therapeutic candidate in diseases such as stroke where inflammation is a central hallmark.

Abstract Background DHCR24, involved in the de novo synthesis of cholesterol and protection of neuronal cells against different stress conditions, has been shown to be selectively downregulated in neurons of the affected brain areas in Alzheimer’s disease. Methods Here, we investigated whether the overexpression of DHCR24 protects neurons against inflammation-induced neuronal death using co-cultures of mouse embryonic primary cortical neurons and BV2 microglial cells upon acute neuroinflammation. Moreover, the effects of DHCR24 overexpression on dendritic spine density and morphology in cultured mature mouse hippocampal neurons and on the outcome measures of ischemia-induced brain damage in vivo in mice were assessed. Results Overexpression of DHCR24 reduced the loss of neurons under inflammation elicited by LPS and IFN-γ treatment in co-cultures of mouse neurons and BV2 microglial cells but did not affect the production of neuroinflammatory mediators, total cellular cholesterol levels, or the activity of proteins linked with neuroprotective signaling. Conversely, the levels of post-synaptic cell adhesion protein neuroligin-1 were significantly increased upon the overexpression of DHCR24 in basal growth conditions. Augmentation of DHCR24 also increased the total number of dendritic spines and the proportion of mushroom spines in mature mouse hippocampal neurons. In vivo, overexpression of DHCR24 in striatum reduced the lesion size measured by MRI in a mouse model of transient focal ischemia. Conclusions These results suggest that the augmentation of DHCR24 levels provides neuroprotection in acute stress conditions, which lead to neuronal loss in vitro and in vivo.

Dysregulation of brain iron metabolism is one of the pathological features of aging and Alzheimer’s disease (AD), a neurodegenerative disease characterized by progressive memory loss and cognitive impairment. While physical inactivity is one of the risk factors for AD and regular exercise improves cognitive function and reduces pathology associated with AD, the underlying mechanisms remain unclear. The purpose of the study is to explore the effect of regular physical exercise on modulation of iron homeostasis in the brain and periphery of the 5xFAD mouse model of AD. By using inductively coupled plasma mass spectrometry and a variety of biochemical techniques, we measured total iron content and level of proteins essential in iron homeostasis in the brain and skeletal muscles of sedentary and exercised mice. Long-term voluntary running induced redistribution of iron resulted in altered iron metabolism and trafficking in the brain and increased iron content in skeletal muscle. Exercise reduced levels of cortical hepcidin, a key regulator of iron homeostasis, coupled with interleukin-6 (IL-6) decrease in cortex and plasma. We propose that regular exercise induces a reduction of hepcidin in the brain, possibly via the IL-6/STAT3/JAK1 pathway. These findings indicate that regular exercise modulates iron homeostasis in both wild-type and AD mice.

Neural stem/progenitor cells (NPCs) generate new neurons in the brain throughout an individual's lifetime in an intricate process called neurogenesis. Neurogenic alterations are a common feature of several adult-onset neurodegenerative diseases. The neuronal ceroid lipofuscinoses (NCLs) are the most common group of inherited neurodegenerative diseases that mainly affect children. Pathological features of the NCLs include accumulation of lysosomal storage material, neuroinflammation and neuronal degeneration, yet the exact cause of this group of diseases remains poorly understood. The function of the CLN5 protein, causative of the CLN5 disease form of NCL, is unknown. In the present study, we sought to examine neurogenesis in the neurodegenerative disorder caused by loss of Cln5. Our findings demonstrate a newly identified crucial role for CLN5 in neurogenesis. We report for the first time that neurogenesis is increased in Cln5-deficient mice, which model the childhood neurodegenerative disorder caused by loss of Cln5. Our results demonstrate that, in Cln5 deficiency, proliferation of NPCs is increased, NPC migration is reduced and NPC differentiation towards the neuronal lineage is increased concomitantly with functional alterations in the NPCs. Moreover, the observed impairment in neurogenesis is correlated with increased expression of the pro-inflammatory cytokine IL-1β. A full understanding of the pathological mechanisms that lead to disease and the function of the NCL proteins are critical for designing effective therapeutic approaches for this devastating neurodegenerative disorder.

Abstract The Finnish variant of late infantile neuronal ceroid lipofuscinosis (CLN5 disease) belongs to a family of neuronal ceroid lipofuscinosis (NCLs) diseases. Vision loss is among the first clinical signs in childhood forms of NCLs. Mutations in CLN5 underlie CLN5 disease. The aim of this study was to characterize how the lack of normal functionality of the CLN5 protein affects the mouse retina. Scotopic electroretinography (ERG) showed a diminished c-wave amplitude in the CLN5 deficient mice already at 1 month of age, indicative of pathological events in the retinal pigmented epithelium. A- and b-waves showed progressive impairment later from 2 and 3 months of age onwards, respectively. Structural and immunohistochemical (IHC) analyses showed preferential damage of photoreceptors, accumulation of autofluorescent storage material, apoptosis of photoreceptors, and strong inflammation in the CLN5 deficient mice retinas. Increased levels of autophagy-associated proteins Beclin-1 and P62, and increased LC3b-II/LC3b-I ratio, were detected by Western blotting from whole retinal extracts. Photopic ERG, visual evoked potentials, IHC and cell counting indicated relatively long surviving cone photoreceptors compared to rods. In conclusion, CLN5 deficient mice develop early vision loss that reflects the condition reported in clinical childhood forms of NCLs. The vision loss in CLN5 deficient mice is primarily caused by photoreceptor degeneration.

Extracellular ATP activates inflammasome and triggers the release of multiple cytokines in various immune cells, a process primarily mediated by P2X7 receptors. However, the expression and functional properties of P2X7 receptors in native mast cells in tissues such as meninges where migraine pain originates from have not been explored. Here we report a novel model of murine cultured meningeal mast cells and using these, as well as easily accessible peritoneal mast cells, studied the mechanisms of ATP-mediated mast cell activation. We show that ATP induced a time and dose-dependent activation of peritoneal mast cells as analyzed by the uptake of organic dye YO-PRO1 as well as 4,6-diamidino-2-phenylindole (DAPI). Both YO-PRO1 and DAPI uptake in mast cells was mediated by the P2X7 subtype of ATP receptors as demonstrated by the inhibitory effect of P2X7 antagonist A839977. Consistent with this, significant YO-PRO1 uptake was promoted by the P2X7 agonist 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP). Extracellular ATP-induced degranulation of native and cultured meningeal mast cells was shown with Toluidine Blue staining. Taken together, these data demonstrate the important contribution of P2X7 receptors to ATP-driven activation of mast cells, suggesting these purinergic mechanisms as potential triggers of neuroinflammation and pain sensitization in migraine.

Microglia are resident immune cells of the central nervous system and play critical roles during the development, homeostasis, and pathologies of the brain. Originated from yolk sac erythromyeloid progenitors, microglia immigrate into the embryonic brain parenchyma to undergo final postnatal differentiation and maturation driven by distinct chemokines, cytokines, and growth factors. Among them, TGFβ1 is an important regulator of microglial functions, mediating homeostasis, anti-inflammation, and triggering the expression of microglial homeostatic signature genes. Since microglia studies are mainly based on rodent cells and the isolation of homeostatic microglia from human tissue is challenging, human-induced pluripotent stem cells have been successfully differentiated into microglia-like cells recently. However, employed differentiation protocols strongly vary regarding used cytokines and growth factors, culture conditions, time span, and cell yield. Moreover, the incomplete differentiation of human microglia can hamper the similarity to primary human microglia and dramatically influence the outcome of follow-up studies with these differentiated cells. This review summarizes the current knowledge of the molecular mechanisms driving rodent microglia differentiation in vivo, further compares published differentiation protocols, and highlights the potential of TGFβ as an essential maturation factor.

Microglia are involved in the post-stroke immunomodulation of brain plasticity, repair, and reorganization. Here, we evaluated whether adipose-tissue-derived mesenchymal stem cells (ADMSCs) and/or rehabilitation improve behavioral recovery by modulating long-term perilesional inflammation and creating a recovery-permissive environment in a rat model of ischemic stroke. Methods: A two-way mixed lymphocyte reaction was used to assess the immunomodulatory capacity of ADMSCs in vitro. Two or 7 days after permanent middle cerebral artery occlusion (pMCAO), rats were intravenously administered ADMSCs or vehicle and housed in a standard or enriched environment (EE). Behavioral performance was assessed with a cylinder test, then we performed stereological and ImageJ/Fiji quantifications of ionized calcium-binding adaptor molecule 1 (Iba1) cells and blood–brain barrier (BBB) leakage. Results: Human ADMSCs were immunosuppressive in vitro. The cylinder test showed partial spontaneous behavioral recovery of pMCAO rats, which was further improved by combined ADMSCs and housing in EE on days 21 and 42 (p < 0.05). We detected an ischemia-induced increase in numbers, staining intensity, and branch length of Iba1+ microglia/macrophages as well as BBB leakage in the perilesional cortex. However, these were not different among pMCAO groups. Conclusion: Combined cell therapy and rehabilitation additively improved behavioral outcome despite long-term perilesional microglia presence in stroke rats.

Background: Neuroinflammation and biometal dyshomeostasis are key pathological features of several neurodegenerative diseases, including Alzheimer’s disease (AD). Inflammation and biometals are linked at the molecular level through regulation of metal buffering proteins such as the metallothioneins. Even though the molecular connections between metals and inflammation have been demonstrated, little information exists on the effect of copper modulation on brain inflammation.Methods: We demonstrate the immunomodulatory potential of the copper bis(thiosemicarbazone) complex CuII(atsm) in an neuroinflammatory model in vivo and describe its anti-inflammatory effects on microglia and astrocytes in vitro.Results: By using a sophisticated in vivo magnetic resonance imaging (MRI) approach, we report the efficacy of CuII(atsm) in reducing acute cerebrovascular inflammation caused by peripheral administration of bacterial lipopolysaccharide (LPS). CuII(atsm) also induced anti-inflammatory outcomes in primary microglia [significant reductions in nitric oxide (NO), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor (TNF)] and astrocytes [significantly reduced NO, MCP-1, and interleukin 6 (IL-6)] in vitro. These anti-inflammatory actions were associated with increased cellular copper levels and increased the neuroprotective protein metallothionein-1 (MT1) in microglia and astrocytes.Conclusion: The beneficial effects of CuII(atsm) on the neuroimmune system suggest copper complexes are potential therapeutics for the treatment of neuroinflammatory conditions.

Objectives: Immune responses in migraine are poorly characterized, yet implicated in the disease pathogenesis. This study was carried out to characterize purinergic profiles of T-cells in patients with episodic migraine without aura (MWoA) to provide mechanistic evidence for ATP and adenosine involvement in modulation of immune regulation in migraine.Methods: Peripheral blood samples were obtained from patients with migraine (n = 16) and age-matched control subjects (n = 21). Subsets of T-cells were identified by flow cytometry based on specific membrane markers.Results: Migraine patients showed reduced total T-cell counts in the peripheral blood. Whereas the total number of CD3+CD4+, CD3+CD8+, or regulatory T lymphocytes (Treg) was not changed, the proportion of Treg CD45R0+CD62L– and CD45R0–CD62L– cells was increased. Interestingly, in migraine, less Treg cells expressed CD39 and CD73 suggesting disrupted ATP breakdown to adenosine. The negative correlations were observed between the duration of migraine and the relative number of CD73+CD39– Tregs and total number of CD73-positive CD45R0+CD62L+ Tregs.Conclusion: Obtained data indicate that T-cell populations are altered in episodic migraine and suggest the involvement of Tregs in the pathophysiology of this disorder. Reduced expression of CD39 and CD73 suggests promotion of ATP-dependent pro-inflammatory and reduction of adenosine-mediated anti-inflammatory mechanisms in migraine.

Nuclear receptors have generated substantial interest in the past decade as potential therapeutic targets for the treatment of neurodegenerative disorders. Despite years of effort, effective treatments for progressive neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and ALS remain elusive, making non-classical drug targets such as nuclear receptors an attractive alternative. A substantial literature in mouse models of disease and several clinical trials have investigated the role of nuclear receptors in various neurodegenerative disorders, most prominently AD. These studies have met with mixed results, yet the majority of studies in mouse models report positive outcomes. The mechanisms by which nuclear receptor agonists affect disease pathology remain unclear. Deciphering the complex signaling underlying nuclear receptor action in neurodegenerative diseases is essential for understanding this variability in preclinical studies, and for the successful translation of nuclear receptor agonists into clinical therapies.

Inflammation is a major mechanism of acute brain injury and chronic neurodegeneration. This neuroinflammation is known to be substantially regulated by the transcription factor NF-κB, which is predominantly found in the form of heterodimer of p65 (RelA) and p50 subunit, with p50/p50 homodimers being also common. The p65 subunit has a transactivation domain, whereas p50 is chiefly involved in DNA binding. Binding of the p65/p50 heterodimers is thought to induce expression of numerous proinflammatory genes in microglia. Here we show that cultured microglia deficient for the gene (Nfkb1) encoding p50 subunit show reduced induction of proinflammatory mediators, increased expression of anti-inflammatory genes, and increased expression of CD45, an immunoregulatory molecule, in response to lipopolysaccharide (LPS) exposure, but increased capacity to take up β-amyloid (Aβ) which is associated with enhanced release of tumor necrosis factor alpha (TNFα). However, Nfkb1 deficiency strongly increases leukocyte infiltration and the expression of proinflammatory genes in response to intrahippocampal administration of LPS. Also, when crossing Nfkb1 deficient mice with APdE9 transgenic mice the expression of proinflammatory genes was strongly enhanced, whereas Aβ burden was slightly but significantly reduced. These alterations in expression of inflammatory mediators in Nfkb1 deficient mice were associated with reduced expression of CD45. Our data demonstrates a crucial and complex role p50 subunit of NF-κB in brain inflammation, especially in regulating the phenotype of microglia after acute and chronic inflammatory insults relevant to clinical conditions, contributing to both pro-inflammatory and anti-inflammatory responses of microglia, infiltration of leukocytes, and clearance of Aβ in Alzheimer's disease.