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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.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with short life expectancy and no effective therapy. We previously identified upregulated miR-124 in NSC-34-motor neurons (MNs) expressing human SOD1-G93A (mSOD1) and established its implication in mSOD1 MN degeneration and glial cell activation. When anti-miR-124-treated mSOD1 MN (preconditioned) secretome was incubated in spinal cord organotypic cultures from symptomatic mSOD1 mice, the dysregulated homeostatic balance was circumvented. To decipher the therapeutic potential of such preconditioned secretome, we intrathecally injected it in mSOD1 mice at the early stage of the disease (12-week-old). Preconditioned secretome prevented motor impairment and was effective in counteracting muscle atrophy, glial reactivity/dysfunction, and the neurodegeneration of the symptomatic mSOD1 mice. Deficits in corticospinal function and gait abnormalities were precluded, and the loss of gastrocnemius muscle fiber area was avoided. At the molecular level, the preconditioned secretome enhanced NeuN mRNA/protein expression levels and the PSD-95/TREM2/IL-10/arginase 1/MBP/PLP genes, thus avoiding the neuronal/glial cell dysregulation that characterizes ALS mice. It also prevented upregulated GFAP/Cx43/S100B/vimentin and inflammatory-associated miRNAs, specifically miR-146a/miR-155/miR-21, which are displayed by symptomatic animals. Collectively, our study highlights the intrathecal administration of the secretome from anti-miR-124-treated mSOD1 MNs as a therapeutic strategy for halting/delaying disease progression in an ALS mouse model., This research was funded by Santa Casa da Misericórdia de Lisboa: ELA-2015-002 (to DB); Fundação para a Ciência e a Tecnologia (FCT): PTDC/MED-NEU/31395/2017 (to D.B.), UIDB/UIDP/04138/2020, and UID/DTP/04138/2019-2020 (to iMed.ULisboa); Programa Operacional Regional de Lisboa and the Programa Operacional Competitividade e Internacionalização LISBOA-01-0145-FEDER-031395 (to D.B.); La Caixa Foundation and Francisco Luzón Foundation through project HR21-00931 (to D.B.); and an individual fellowship from FCT: SFRH/BD/129586/2017 (to M.B.). This work was also funded by the ICVS Scientific Microscopy Platform, a member of the national infrastructure of PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122).

Microglia-associated inflammation and miRNA dysregulation are key players in Alzheimer’s disease (AD) pathophysiology. Previously, we showed miR-124 upregulation in APP Swedish SH-SY5Y (SWE) and PSEN1 iPSC-derived neurons and its propagation by the secretome (soluble and exosomal fractions). After modulation with miR-124 mimic/inhibitor, we identified common responsive mechanisms between such models. We also reported miR-124 colocalization with microglia in AD patient hippocampi. Herein, we determined how miR-124 modulation in SWE cells influences microglia polarized subtypes in the context of inflammation. We used a coculture system without cell-to-cell contact formed by miR-124 modulated SWE cells and human CHME3 microglia stimulated with interferon-gamma (IFNγ-MG), in which we assessed their adopted gene/miRNA profile and proteomic signature. The increase of miR-124 in SWE cells/secretome (soluble and exosomal) was mimicked in IFNγ-MG. Treatment of SWE cells with the miR-124 inhibitor led to RAGE overexpression and loss of neuronal viability, while the mimic caused RAGE/HMGB1 downregulation and prevented mitochondria membrane potential loss. When accessing the paracrine effects on microglia, SWE miR-124 inhibitor favored their IFNγ-induced inflammatory signature (upregulated RAGE/HMGB1/iNOS/IL-1β; downregulated IL-10/ARG-1), while the mimic reduced microglia activation (downregulated TNF-α/iNOS) and deactivated extracellular MMP-2/MMP-9 levels. Microglia proteomics identified 113 responsive proteins to SWE miR-124 levels, including a subgroup of 17 proteins involved in immune function/inflammation and/or miR-124 targets. A total of 72 proteins were downregulated (e.g., MAP2K6) and 21 upregulated (e.g., PAWR) by the mimic, while the inhibitor also upregulated 21 proteins and downregulated 17 (e.g., TGFB1, PAWR, and EFEMP1). Other targets were associated with neurodevelopmental mechanisms, synaptic function, and vesicular trafficking. To examine the source of miR-124 variations in microglia, we silenced the RNase III endonuclease Dicer1 to block miRNA canonical biogenesis. Despite this suppression, the coculture with SWE cells/exosomes still raised microglial miR-124 levels, evidencing miR-124 transfer from neurons to microglia. This study is pioneer in elucidating that neuronal miR-124 reshapes microglia plasticity and in revealing the relevance of neuronal survival in mechanisms underlying inflammation in AD-associated neurodegeneration. These novel insights pave the way for the application of miRNA-based neuropharmacological strategies in AD whenever miRNA dysregulated levels are identified during patient stratification.

As aging and cognitive decline progresses, the impact of a sedentary lifestyle on the appearance of environment-dependent cellular morphologies in the brain becomes more apparent. Sedentary living is also associated with poor oral health, which is known to correlate with the rate of cognitive decline. Here, we will review the evidence for the interplay between mastication and environmental enrichment and assess the impact of each on the structure of the brain. In previous studies, we explored the relationship between behavior and the morphological features of dentate gyrus glial fibrillary acidic protein (GFAP)-positive astrocytes during aging in contrasting environments and in the context of induced masticatory dysfunction. Hierarchical cluster and discriminant analysis of GFAP-positive astrocytes from the dentate gyrus molecular layer revealed that the proportion of AST1 (astrocyte arbors with greater complexity phenotype) and AST2 (lower complexity) are differentially affected by environment, aging and masticatory dysfunction, but the relationship is not straightforward. Here we re-evaluated our previous reconstructions by comparing dorsal and ventral astrocyte morphologies in the dentate gyrus, and we found that morphological complexity was the variable that contributed most to cluster formation across the experimental groups. In general, reducing masticatory activity increases astrocyte morphological complexity, and the effect is most marked in the ventral dentate gyrus, whereas the effect of environment was more marked in the dorsal dentate gyrus. All morphotypes retained their basic structural organization in intact tissue, suggesting that they are subtypes with a non-proliferative astrocyte profile. In summary, the increased complexity of astrocytes in situations where neuronal loss and behavioral deficits are present is counterintuitive, but highlights the need to better understand the role of the astrocyte in these conditions.

N-Ethylhexedrone (NEH) and buphedrone (Buph) are emerging synthetic cathinones (SC) with limited information about their detrimental effects within central nervous system. Objectives: To distinguish mice behavioural changes by NEH and Buph and validate their differential harmful impact on human neurons and microglia. In vivo safety data showed the typical induced behaviour of excitation and stereotypies with 4-64 mg/kg, described for other SC. Buph additionally produced jumping and aggressiveness signs, while NEH caused retropulsion and circling. Transient reduction in body-weight gain was obtained with NEH at 16 mg/kg and induced anxiolytic-like behaviour mainly with Buph. Both drugs generated place preference shift in mice at 4 and 16 mg/kg, suggestive of abuse potential. In addition, mice withdrawn NEH displayed behaviour suggestive of depression, not seen with Buph. When tested at 50-400 μM in human nerve cell lines, NEH and Buph caused neuronal viability loss at 100 μM, but only NEH produced similar results in microglia, indicating different cell susceptibilities. NEH mainly induced microglial late apoptosis/necrosis, while Buph caused early apoptosis. NEH was unique in triggering microglia shorter/thicker branches indicative of cell activation, and more effective in increasing microglial lysosomal biogenesis (100 μM vs. 400 μM Buph), though both produced the same effect on neurons at 400 μM. These findings indicate that NEH and Buph exert neuro-microglia toxicities by distinct mechanisms and highlight NEH as a specific inducer of microglia activation. Buph and NEH showed in vivo/in vitro neurotoxicities but enhanced specific NEH-induced behavioural and neuro-microglia dysfunctionalities pose safety concerns over that of Buph.

Microglia are CNS-resident cells involved in immune surveillance and maintenance of intercellular homeostasis, while also contributing to neurologic pathologies. MicroRNAs (miRNAs) are a class of small (~22 nucleotides) single-stranded noncoding RNAs that participate in gene regulation at the post-transcriptional level. miRNAs typically bind to the untranslated region (3' UTR) of RNAs. It has been shown that miRNAs are important players in controlling inflammation and that their abnormal expression is linked to cancer and ageing, and to the onset and progression of neurodegenerative disease. Furthermore, miRNAs participate in intercellular trafficking. Thus, miRNAs are released from cells in a free form, bound to proteins or packaged within extracellular vesicles (EVs), exerting paracrine and long distance signaling. In this review, recent findings on the role of miRNAs as drivers of microglia phenotypic changes and their cotribution in neurological disease are addressed. MAIN POINTS: miRNAs have a key role in microglia function/dysfunction, polarization, and restoration. Microglia are both a source and recipient of extracellular vesicles (EVs) containing miRNAs. Extracellular miRNAs may be found as soluble (free and EV cargo) and protein complexes.

Studies have correlated excessive S100B, a small inflammatory molecule, with demyelination and associated inflammatory processes occurring in multiple sclerosis. The relevance of S100B in multiple sclerosis pathology brought an emerging curiosity highlighting its use as a potential therapeutic target to reduce damage during the multiple sclerosis course, namely during inflammatory relapses. We examined the relevance of S100B and further investigated the potential of S100B-neutralizing small-molecule pentamidine in chronic experimental autoimmune encephalomyelitis. S100B depletion had beneficial pathological outcomes and based on promising results of a variety of S100B blockade strategies in an ex vivo demyelinating model, we choose pentamidine to assay its role in the in vivo experimental autoimmune encephalomyelitis. We report that pentamidine prevents more aggressive clinical symptoms and improves recovery of chronic experimental autoimmune encephalomyelitis. Blockade of S100B by pentamidine protects against oligodendrogenesis impairment and neuroinflammation by reducing astrocyte reactivity and microglia pro-inflammatory phenotype. Pentamidine also increased regulatory T cell density in the spinal cord suggesting an additional immunomodulatory action. These results showed the relevance of S100B as a main driver of neuroinflammation in experimental autoimmune encephalomyelitis and identified an uncharacterized mode of action of pentamidine, strengthening the possibility to use this drug as an anti-inflammatory and remyelinating therapy for progressive multiple sclerosis.

Small extracellular vesicles (sEVs) have ∼30–200 nm diameter size and may act as carriers of different cargoes, depending on the cell of origin or on the physiological/pathological condition. As endogenous nanovesicles, sEVs are important in intercellular communication and have many of the desirable features of an ideal drug delivery system. sEVs are naturally biocompatible, with superior targeting capability, safety profile, nanometric size, and can be loaded with both lipophilic and hydrophilic agents. Because of their biochemical and physical properties, sEVs are considered a promising strategy over other delivery vehicles in the central nervous system (CNS) since they freely cross the blood-brain barrier and they can be directed to specific nerve cells, potentiating a more precise targeting of their cargo. In addition, sEVs remain stable in the peripheral circulation, making them attractive nanocarrier systems to promote neuroregeneration. This review focuses on the recent progress in methods for manufacturing, isolating, and engineering sEVs that can be used as a therapeutic strategy to overcome neurodegeneration associated with pathologies of the CNS, with particular emphasis on Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis diseases, as well as on brain tumors.

At birth, human neonates are more likely to develop cholestasis and oxidative stress due to immaturity or other causes. We aimed to search for a potential association between bile acids profile, redox status, and type of diet in healthy infants.A cross-sectional, exploratory study enrolled 2-month-old full-term infants (n = 32). We measured plasma bile acids (total and conjugated), and red blood cell (RBC) oxidative stress biomarkers. The type of diet (breastfeeding, mixed, formula) was used as an independent variable.Plasma total bile acids medium value was 14.80 µmol/L (IQR: 9.25-18.00). The plasma-conjugated chenodeoxycholic acid percentage (CDCA%) correlated significantly and negatively with RBCs membrane-bound hemoglobin percentage (MBH%) (r = -0.635, p 0.01) and with RBC-oxidized glutathione (r = -0.403, p 0.05) levels. RBC oxidative stress biomarkers (especially MBH%) were predictors of conjugated CDCA%, and this predictive ability was enhanced when adjusted for the type of diet (MBH, r = 0.452, p 0.001).Our data suggest that the bile acid profile might play a role in the regulation of redox status (or vice versa) in early postnatal life. Eventually, the type of diet may have some impact on this process.The conjugated CDCA% in plasma is negatively correlated with biomarkers of RBC oxidative stress in healthy infants. Specific biomarkers of RBC oxidative stress (e.g. MBH, GSH, GSSG) may be promising predictors of conjugated CDCA% in plasma. The type of diet may influence the predictive ability of hit RBC oxidative stress biomarkers (e.g. MBH, GSH, GSSG). Our findings suggest a link between plasma bile acids profile and the RBC redox status in healthy infants, eventually modulated by the type of diet. The recognition of this link may contribute to the development of preventive and therapeutic strategies for neonatal cholestasis.

Neuronal miRNA dysregulation may have a role in the pathophysiology of Alzheimer’s disease (AD). miRNA(miR)-124 is largely abundant and a critical player in many neuronal functions. However, the lack of models reliably recapitulating AD pathophysiology hampers our understanding of miR-124’s role in the disease. Using the classical human SH-SY5Y-APP695 Swedish neuroblastoma cells (SH-SWE) and the PSEN1 mutant iPSC-derived neurons (iNEU-PSEN), we observed a sustained upregulation of miR-124/miR-125b/miR-21, but only miR-124 was consistently shuttled into their exosomes. The miR-124 mimic reduced APP gene expression in both AD models. While miR-124 mimic in SH-SWE neurons led to neurite outgrowth, mitochondria activation and small Aβ oligomer reduction, in iNEU-PSEN cells it diminished Tau phosphorylation, whereas miR-124 inhibitor decreased dendritic spine density. In exosomes, cellular transfection with the mimic predominantly downregulated miR-125b/miR-21/miR-146a/miR-155. The miR-124 inhibitor upregulated miR-146a in the two experimental cell models, while it led to distinct miRNA signatures in cells and exosomes. In sum, though miR-124 function may be dependent on the neuronal AD model, data indicate that keeping miR-124 level strictly controlled is crucial for proper neuronal function. Moreover, the iNEU-PSEN cellular model stands out as a useful tool for AD mechanistic studies and perhaps for the development of personalized therapeutic strategies.

Supplementary material of the manuscript entitled: "Neuronal dynamics and miRNA signaling differ in SH-SY5Y APPSwe and PSEN1 mutant iPSC-derived AD models by miR-124 mimic and inhibitor modulation"

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder, characterized by the degeneration of upper and lower motor neurons of the motor cortex, brainstem, and ventral horn of the spinal cord. The role of glial cells in the onset and progression of ALS is increasingly being recognized. Dysfunctional astrocytes, with an atypical and neurotoxic phenotype, in the cerebral cortex and the spinal cord promote neuroinflammation and motor neuron degeneration. Indeed, cortical and spinal cord astrocytes from SOD1G93A (mSOD1) mice are neurotoxic, develop early deficits, and lose their neuro-supportive properties before disease onset. This chapter discusses the contribution of dysfunctional cortical and spinal cord astrocytes in the development and progression of ALS. Differences in astrocyte heterogeneity and reactivity, calcium signaling, neurotransmitters, and in paracrine signaling mechanisms along with implications for novel therapies in ALS are addressed.

A lack of stratification methods in patients with amyotrophic lateral sclerosis (ALS) is likely implicated in therapeutic failures. Regional diversities and pathophysiological abnormalities in astrocytes from mice with SOD1 mutations (mSOD1-ALS) can now be explored in human patients using somatic cell reprogramming. Here, fibroblasts from four sporadic (sALS) and three mSOD1-ALS patients were transdifferentiated into induced astrocytes (iAstrocytes). ALS iAstrocytes were neurotoxic toward HB9-GFP mouse motor neurons (MNs) and exhibited subtype stratification through GFAP, CX43, Ki-67, miR-155 and miR-146a expression levels. Up- (two cases) and down-regulated (three cases) miR-146a values in iAstrocytes were recapitulated in their secretome, either free or as cargo in small extracellular vesicles (sEVs). We previously showed that the neuroprotective phenotype of depleted miR-146 mSOD1 cortical astrocytes was reverted by its mimic. Thus, we tested such modulation in the most miR-146a-depleted patient-iAstrocytes (one sALS and one mSOD1-ALS). The miR-146a mimic in ALS iAstrocytes counteracted their reactive/inflammatory profile and restored miR-146a levels in sEVs. A reduction in lysosomal activity and enhanced synaptic/axonal transport-related genes in NSC-34 MNs occurred after co-culture with miR-146a-modulated iAstrocytes. In summary, the regulation of miR-146a in depleted ALS astrocytes may be key in reestablishing their normal function and in restoring MN lysosomal/synaptic dynamic plasticity in disease sub-groups.

Brazilian Research Council CNPq (grant number 475677/2008-0) Funda??o Amaz?nia Paraense de Amparo ? Pesquisa (FAPESPA, grant number 136/08). Funda??o de Amparo e Desenvolvimento da Pesquisa (FADESP) and the Pr?-Reitoria de Pesquisa e P?s-Gradua??o (PROPESP/UFPA) Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES). Programa Nacional de Coopera??o Acad?mica da Amaz?nia ? DRI (Processo CAPES No. 88881.370785/2019-01). Funda??o para a Ci?ncia e a Tecnologia (PTDC/MED-NEU/31395/2017, LISBOA-01-0145-FEDER031395, and UID/DTP/04138/2018-2021), Santa Casa da Miseric?rdia (ALS Research Grant). Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Hospital Universit?rio Jo?o de Barros Barreto. Laborat?rio de Investiga??es em Neurodegenera??o e Infec??o. Bel?m, PA, Brazil / Centro Universit?rio do Estado do Par?. Curso de Medicina. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Hospital Universit?rio Jo?o de Barros Barreto. Laborat?rio de Investiga??es em Neurodegenera??o e Infec??o. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Hospital Universit?rio Jo?o de Barros Barreto. Laborat?rio de Investiga??es em Neurodegenera??o e Infec??o. Bel?m, PA, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Microscopia Eletr?nica. Ananindeua, PA, Brasil. University of Oxford. Department of Pharmacology. Laboratory of Experimental Neuropathology. Oxford, United Kingdom. Universidade de Lisboa. Faculty of Pharmacy. Research Institute for Medicines. Lisbon, Portugal / Universidade de Lisboa. Faculty of Pharmacy. Department of Biochemistry and Human Biology. Lisbon, Portugal. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Hospital Universit?rio Jo?o de Barros Barreto. Laborat?rio de Investiga??es em Neurodegenera??o e Infec??o. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Hospital Universit?rio Jo?o de Barros Barreto. Laborat?rio de Investiga??es em Neurodegenera??o e Infec??o. Bel?m, PA, Brazil. To explore the impact of reduced mastication and a sedentary lifestyle on spatial learning and memory in the aged mice, as well as on the morphology of astrocytes in the molecular layer of dentate gyrus (MolDG), different masticatory regimens were imposed. Control mice received a pellet-type hard diet, while the reduced masticatory activity group received a pellet diet followed by a powdered diet, and the masticatory rehabilitation group received a pellet diet, followed by powder diet and then a pellet again. To mimic sedentary or active lifestyles, mice were housed in an impoverished environment of standard cages or in an enriched environment. The Morris Water Maze (MWM) test showed that masticatory-deprived group, regardless of environment, was not able to learn and remember the hidden platform location, but masticatory rehabilitation combined with enriched environment recovered such disabilities. Microscopic three-dimensional reconstructions of 1,800 glial fibrillary acidic protein (GFAP)-immunolabeled astrocytes from the external third of the MolDG were generated using a stereological systematic and random sampling approach. Hierarchical cluster analysis allowed the characterization into two main groups of astrocytes with greater and lower morphological complexities, respectively, AST1 and AST2. When compared to the hard diet group subjected to impoverished environment, deprived animals maintained in the same environment for 6 months showed remarkable shrinkage of astrocyte branches. However, the long-term environmental enrichment (18-monthold) applied to the deprived group reversed the shrinkage effect, with significant increase in the morphological complexity of AST1 and AST2, when in an impoverished or enriched environment. During housing under enriched environment, complexity of branches of AST1 and AST2 was reduced by the powder diet (pellet followed by powder regimes) in young but not in old mice, where it was reversed by pellet diet (pellet followed by powder and pellet regime again). The same was not true for mice housed under impoverished environment. Interestingly, we were unable to find any correlation between MWM data and astrocyte morphological changes. Our findings indicate that both young and aged mice subjected to environmental enrichment, and under normal or rehabilitated masticatory activity, preserve spatial learning and memory. Nonetheless, data suggest that an impoverished environment and reduced mastication synergize to aggravate age-related cognitive decline; however, the association with morphological diversity of AST1 and AST2 at the MolDG requires further investigation.

The prevalence of Alzheimer’s disease (AD), the most common cause of age-associated dementia, is estimated to increase over the next decades. Evidence suggests neuro-immune signaling deregulation and risk genes beyond the amyloid-β (Aβ) deposition in AD pathology. We examined the temporal profile of inflammatory mediators and microglia deactivation/activation in the brain cortex and hippocampus of 3xTg-AD mice at 3- and 9-month-old. We found upregulated APP processing, decreased expression of CD11b, CX3CR1, MFG-E8, TNF-α, IL-1β, MHC-II and C/EBP-α and increased miR-146a in both brain regions in 3-month-old 3xTG-AD mice, suggestive of a restrictive regulation. Enhanced TNF-α, IL-1β, IL-6, iNOS, SOCS1 and Arginase 1 were only present in the hippocampus of 9-month-old animals, though elevation of HMGB1 and reduction of miR-146a and miR-124 were common features in the hippocampus and cortex regions. miR-155 increased early in the cortex and later in both regions, supporting its potential as a biomarker. Candidate downregulated target genes by cortical miR-155 included Foxo3, Runx2 and CEBPβ at 3 months and Foxo3, Runx2 and Socs1 at 9 months, which are implicated in cell survival, but also in Aβ pathology and microglia/astrocyte dysfunction. Data provide new insights across AD state trajectory, with divergent microglia phenotypes and inflammatory-associated features, and identify critical targets for drug discovery and combinatorial therapies.

Exosome-mediated intercellular communication has been increasingly recognized as having a broad impact on Alzheimer's disease (AD) pathogenesis. Still, limited information exists regarding their “modus operandi”, as it critically depends on exosomal cargo, environmental context and target cells. Therefore, a more thorough understanding of the role of exosomes from different cell types as mediators of neuroinflammation in AD context is a decisive step to open avenues for innovative and efficient therapies. In this study, we demonstrate that SH-SY5Y cells transfected with the Swedish mutant of APP695 (SHSwe) remarkably express increased inflammatory markers, combined with higher APP and Aβ1-40 production, when compared to naive SH-SY5Y (SH) cells. Although exerting an early clearance effect on extracellular APP and Aβ accumulation when in co-culture with SHSwe cells, human CHME3 microglia gradually lose such property, and express both pro-inflammatory (iNOS, IL-1β, TNF-α, MHC class II, IL-6) and pro-resolving genes (IL-10 and Arginase 1), while also evidence increased senescence-associated β-galactosidase activity. Interestingly, upregulation of inflammatory-associated miRNA (miR)-155, miR-146a and miR-124 by SHSwe secretome shows to be time-dependent and to inversely correlate with their respective targets (SOCS-1, IRAK1 and C/EBP-α). We report that microglia also internalize exosomes released from SHSwe cells, which are enriched in miR-155, miR-146a, miR-124, miR-21 and miR-125b and recapitulate the cells of origin. Furthermore, we show that SHSwe-derived exosomes are capable of inducing acute and delayed microglial upregulation of TNF-α, HMGB1 and S100B pro-inflammatory markers, from which only S100B is found on their derived exosomes. Most importantly, our data reveal that miR-21 is a consistent biomarker that is found not only in SHSwe cells and in their released exosomes, but also in the recipient CHME3 microglia and derived exosomes. This work contributes to the increased understanding of neuron-microglia communication and exosome-mediated neuroinflammation in AD, while highlights miR-21 as a promising biomarker/target for therapeutic intervention.

High levels of the inflammatory molecule S100B protein have been identified in sera from several perinatal inflammatory conditions involving myelin damage and associated with an adverse prognosis or the emergence of sequelea. S100B is essential for oligodendrocyte (OL) differentiation and maturation, but it remains to be established if excessive levels of released S100B upon early brain injury are deleterious in the neurodevelopmental period. Here, we investigated this possibility by evaluating how elevated S100B affects oligodendrogenesis during this period. First, using primary cultures of OL we observed that damage-induced micromolar levels of S100B impair OL differentiation process. S100B elevated concentrations reduced both transition from immature NG2+ oligodendrocyte precursor cells (OPC) to mature MBP+ OL, and morphological maturation of differentiated OL. Interestingly, these effects were abolished by the use of receptor for advanced glycation end-products (RAGE) antagonist FPS-ZM1, suggesting an involvement of the S100B-RAGE axis on oligodendrogenesis impairment. Next, we used organotypic cerebellar slice cultures to explore the role of S100B in a more complex multicellular environment. Also in this model excessive S100B levels impaired oligodendrogenesis resulting in a reduced myelination. Further, elevated S100B levels compromised neuronal and synaptic integrity, while inducing astrogliosis, nuclear factor (NF)-kB activation and inflammation. Again, the FPS-ZM1 co-treatment prevented S100B-induced damaging effects. Overall, our results indicate that persistently elevated S100B levels have deleterious effects during the neurodevelopmental period through RAGE-dependent processes. Thus, targeting high S100B levels and/or S100B-RAGE interaction may constitute good therapeutic strategies to reduce brain injury, including deficits in neuronal architecture, synaptogenesis and myelination associated with perinatal inflammatory conditions.

Purpose: Epidemiological and intervention studies have attempted to link the health effects of a diet rich in fruits and vegetables with the consumption of polyphenols and their impact in neurodegenerative diseases. Studies have shown that polyphenols can cross the intestinal barrier and reach concentrations in the bloodstream able to exert effects in vivo. However, the effective uptake of polyphenols into the brain is still regarded with some reservations. Here we describe a combination of approaches to examine the putative transport of blackberry-digested polyphenols (BDP) across the blood–brain barrier (BBB) and ultimate evaluation of their neuroprotective effects. Methods: BDP was obtained by in vitro digestion of blackberry extract and BDP major aglycones (hBDP) were obtained by enzymatic hydrolysis. Chemical characterization and BBB transport of extracts were evaluated by LC–MSn. BBB transport and cytoprotection of both extracts was assessed in HBMEC monolayers. Neuroprotective potential of BDP was assessed in NT2-derived 3D co-cultures of neurons and astrocytes and in primary mouse cerebellar granule cells. BDP-modulated genes were evaluated by microarray analysis. Results: Components from BDP and hBDP were shown to be transported across the BBB. Physiologically relevant concentrations of both extracts were cytoprotective at endothelial level and BDP was neuroprotective in primary neurons and in an advanced 3D cell model. The major canonical pathways involved in the neuroprotective effect of BDP were unveiled, including mTOR signaling and the unfolded protein response pathway. Genes such as ASNS and ATF5 emerged as novel BDP-modulated targets. Conclusions: BBB transport of BDP and hBDP components reinforces the health benefits of a diet rich in polyphenols in neurodegenerative disorders. Our results suggest some novel pathways and genes that may be involved in the neuroprotective mechanism of the BDP polyphenol components.

Neuroinflammation is an integral part of the neurodegeneration process inherent to several aging dysfunctions. Within the central nervous system, microglia are the effective immune cells, responsible for neuroinflammatory responses. In this study, raspberries were subjected to in vitro digestion simulation to obtain the components that result from the gastrointestinal (GI) conditions, which would be bioaccessible and available for blood uptake. Both the original raspberry extract and the gastrointestinal bioaccessible (GIB) fraction protected neuronal and microglia cells against H2O2-induced oxidative stress and lipopolysaccharide (LPS)-induced inflammation, at low concentrations. Furthermore, this neuroprotective capacity was independent of intracellular ROS scavenging mechanisms. We show for the first time that raspberry metabolites present in the GIB fraction significantly inhibited microglial pro-inflammatory activation by LPS, through the inhibition of Iba1 expression, TNF-α release and NO production. Altogether, this study reveals that raspberry polyphenols may present a dietary route to the retardation or amelioration of neurodegenerative-related dysfunctions.

Accumulation of mutated superoxide dismutase 1 (mSOD1) in amyotrophic lateral sclerosis (ALS) involves injury to motor neurons (MNs), activation of glial cells and immune unbalance. However, neuroinflammation, besides its detrimental effects, also plays beneficial roles in ALS pathophysiology. Therefore, the targeting of microglia to modulate the release of inflammatory neurotoxic mediators and their exosomal dissemination, while strengthening cell neuroprotective properties, has gained growing interest. We used the N9 microglia cell line to identify phenotype diversity upon the overexpression of wild-type (WT; hSOD1WT) and mutated G93A (hSOD1G93A) protein. To investigate how each transduced cell respond to an inflammatory stimulus, N9 microglia were treated with lipopolysaccharide (LPS). Glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS), known to exert neuroprotective properties, were tested for their immunoregulatory properties. Reduced Fizz1, IL-10 and TLR4 mRNAs were observed in both transduced cells. However, in contrast with hSOD1WT-induced decreased of inflammatory markers, microglia transduced with hSOD1G93A showed upregulation of pro-inflammatory (TNF-α/IL-1β/HMGB1/S100B/iNOS) and membrane receptors (MFG-E8/RAGE). Importantly, their derived exosomes were enriched in HMGB1 and SOD1. When inflammatory-associated miRNAs were evaluated, increased miR-146a in cells with overexpressed hSOD1WT was not recapitulated in their exosomes, whereas hSOD1G93A triggered elevated exosomal miR-155/miR-146a, but no changes in cells. LPS stimulus increased M1/M2 associated markers in the naïve microglia, including MFG-E8, miR-155 and miR-146a, whose expression was decreased in both hSOD1WT and hSOD1G93A cells treated with LPS. Treatment with GUDCA or VS led to a decrease of TNF-α, IL-1β, HMGB1, S100B and miR-155 in hSOD1G93A microglia. Only GUDCA was able to increase cellular IL-10, RAGE and TLR4, together with miR-21, while decreased exosomal miR-155 cargo. Conversely, VS reduced MMP-2/MMP-9 activation, as well as upregulated MFG-E8 and miR-146a, while producing miR-21 shuttling into exosomes. The current study supports the powerful role of overexpressed hSOD1WT in attenuating M1/M2 activation, and that of hSOD1G93A in switching microglia from the steady state into a reactive phenotype with low responsiveness to stimuli. This work further reveals GUDCA and VS as promising modulators of microglia immune response by eliciting common and compound-specific molecular mechanisms that may promote neuroregeneration.

Herein we report novel hybrid compounds based on valproic acid and DNA-alkylating triazene moieties, 1, with therapeutic potential for glioblastoma multiforme chemotherapy. We identified hybrid compounds 1d and 1e to be remarkably more potent against glioma and more efficient in decreasing invasive cell properties than temozolomide and endowed with chemical and plasma stability. In contrast to temozolomide, which undergoes hydrolysis to release an alkylating metabolite, the valproate hybrids showed a low potential to alkylate DNA. Key physicochemical properties align for optimal CNS penetration, highlighting the potential of these effective triazene based-hybrids for enhanced anticancer chemotherapy.

Oligodendrocytes are the myelinating cells of the central nervous system that constitute about 5 to 10% of the total glial population. These cells are responsible for myelin sheath production, which is essential not only for the rapid and efficient conduction of the electrical impulses along the axons, but also for preserving axonal integrity. Oligodendrocytes arise from oligodendrocyte progenitor cells that proliferate and differentiate just before and after birth, under a highly-regulated program. Both oligodendrocytes and their precursors are very susceptible to injury by several mechanisms, including excitotoxic damage, oxidative stress and inflammatory events. In this review, we will cover not only several important aspects of oligodendrocyte development and regulatory mechanisms involved in this process, but also some of the most important pathways of injury associated to oligodendrogenesis. Moreover, we will also address some neurological disorders along life journey that present impairment in oligodendrocyte function and in myelination during neurodevelopment, such as periventricular leukomalacia, hypoxia/ischemia and hyperbilirubinemia that in turn can potentiate the emergence of neurological and neurodegenerative diseases like schizophrenia, multiple sclerosis and Alzheimer's disease.

Astrocytes are major contributors of motor neuron (MN) degeneration in amyotrophic lateral sclerosis (ALS). We investigated whether regional and cell maturation differences influence ALS astrocyte malfunction. Spinal and cortical astrocytes from SOD1G93A (mSOD1) 7-day-old mice were cultured for 5 and 13 days in vitro (DIV). Astrocyte aberrancies predominated in 13DIV cells with region specificity. 13DIV cortical mSOD1 astrocytes showed early morphological changes and a predominant reactive and inflammatory phenotype, while repressed proteins and genes were found in spinal cells. Inflammatory-associated miRNAs, e.g. miR-155/miR-21/miR-146a, were downregulated in the first and upregulated in the later ones. Interestingly, depleted miR-155/miR-21/miR-146a in small extracellular vesicles (sEVs/exosomes) was a common pathological feature. Cortical mSOD1 astrocytes induced late apoptosis and kinesin-1 downregulation in mSOD1 NSC-34 MNs, whereas spinal cells upregulated dynein, while decreased nNOS and synaptic-related genes. Both regional-distinct mSOD1 astrocytes enhanced iNOS gene expression in mSOD1 MNs. We provide information on the potential contribution of astrocytes to ALS bulbar-vs. spinal-onset pathology, local influence on neuronal dysfunction and their shared miRNA-depleted exosome trafficking. These causal and common features may have potential therapeutic implications in ALS. Future studies should clarify if astrocyte-derived sEVs are active players in ALS-related neuroinflammation and glial activation.

Current trend of novel psychoactive substances (NPS) among teenagers is posing new clinical, scientific and forensic societal questions. Synthetic cathinones are among the most consumed groups of NPS appearing on the street market and internet on a regular basis. The properties of these substances change regularly, due to structural modification to circumvent legislation. This practice makes almost impossible to characterize its toxicological profiles on an acceptable time scale, mostly due to the time-consuming experiments that must be held in animal models or human cells by standard methods. Such an issue demands the development of a rapid and inexpensive methodology to be used as a high-throughput screening of cathinones’ toxicity. The yeast Saccharomyces cerevisiae shares highly conserved molecular and cellular mechanisms with human cells and has been used before for pharmacological drugs. In the present work it is proposed to use S. cerevisiae growth curves as a high throughput screening method to profile synthetic cathinones toxicity in a short time scale. The results obtained by S. cerevisiae growth curves analysis were compared to differentiated SH-SY5Y human neuronal cells and similar responses were found. The screening tool methodology has shown able to prioritize the most toxics NPS and can be useful for early warning programs on NPS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) loss. Recent evidences highlight astrocytes as important players in MN death, but the mechanism-based neurotoxicity is still unknown. It is also unclear whether activation of astrocytes in ALS occurs differently in the cerebral cortex and spinal cord. We investigated glial and neuronal alterations in the cortex of SOD1G93A (mSOD1) mice in pre-symptomatic and symptomatic stages. We also characterized astrocytes isolated from the cortex of 7-day-old mSOD1 mice for their aberrancy and MN-induced degenerative effects. In the early stage, we identified a reduction of cell proliferation, NF-kB expression, and of vimentin and micro(miR)-146a expression, suggesting a restrained cortical inflammatory status. However, increased NF-kB expression, cell proliferation, and gene expression of HMGB1, connexin 43 and S100B were distinctive of the symptomatic stage, together with MN loss, downregulated unfold protein response, and decreased expression of synaptic proteins, together with that of miR-125b, miR-21, miR-146a, GFAP, and glutamate transporters. Astrocytes cultured for 13 days in vitro showed comparable NF-kB expression and cell proliferation increase, as well as similar microRNA and gene/protein expression profiles (decreased miR-21, miR-146a, GLT-1 and GFAP, and upregulated HMGB1, S100B and connexin-43), thus sustaining astrocytes as the major contributors of cortical homeostasis deregulation in the symptomatic stage. These reactive astrocytes reduced neurite length and synaptophysin expression in NSC-34/hSOD1WT MN-like cells, and induced mitochondria dysfunction, PSD-95 downregulation, metalloproteinase-9 activation, and late apoptosis in NSC-34/hSOD1G93A cells. Data indicate that astrocytes in mSOD1 mice model acquire early phenotypic aberrancies and highlight downregulated miR-146a as a biomarker and drug target in ALS.

Exosome-mediated intercellular communication has been increasingly recognized as having a broad impact on Alzheimer's disease (AD) pathogenesis. Still, limited information exists regarding their "modus operandi", as it critically depends on exosomal cargo, environmental context and target cells. Therefore, a more thorough understanding of the role of exosomes from different cell types as mediators of neuroinflammation in AD context is a decisive step to open avenues for innovative and efficient therapies. In this study, we demonstrate that SH-SY5Y cells transfected with the Swedish mutant of APP

Bilirubin-induced neurologic dysfunction (BIND) and kernicterus has been used to describe moderate to severe neurologic dysfunction observed in children exposed to excessive levels of total serum bilirubin (TSB) during the neonatal period. Here we use a new mouse model that targets deletion of the Ugt1 locus and the Ugt1a1 gene in liver to promote hyperbilirubinemia-induced seizures and central nervous system toxicity. The accumulation of TSB in these mice leads to diffuse yellow coloration of brain tissue and a marked cerebellar hypoplasia that we characterize as kernicterus. Histologic studies of brain tissue demonstrate that the onset of severe neonatal hyperbilirubinemia, characterized by seizures, leads to alterations in myelination and glia reactivity. Kernicterus presents as axonopathy with myelination deficits at different brain regions, including pons, medulla oblongata, and cerebellum. The excessive accumulation of TSB in the early neonatal period (5 days after birth) promotes activation of the myelin basic protein (Mbp) gene with an accelerated loss of MBP that correlates with a lack of myelin sheath formation. These changes were accompanied by increased astroglial and microglial reactivity, possibly as a response to myelination injury. Interestingly, cerebellum was the area most affected, with greater myelination impairment and glia burden, and showing a marked loss of Purkinje cells and reduced arborization of the remaining ones. Thus, kernicterus in this model displays not only axonal damage but also myelination deficits and glial activation in different brain regions that are usually related to the neurologic sequelae observed after severe hyperbilirubinemia.

Vascular and glial involvement in the development of neurodegenerative disorders, such as Alzheimer's disease (AD), and age-related brain vulnerabilities have been suggested. Therefore, we sought to: (i) investigate which vascular and glial events are evident in ageing and/or AD, (ii) to establish the temporal evolution of vascular and glial changes in AD-like and wild-type (WT) mice and (iii) to relate them to amyloid-β (Aβ) peptide accumulation. We examined immunohistochemically hippocampi and cortex from APP/PS1dE9 and WT C57BL/6 mice along ageing and disease progression (young-adulthood, middle- and old-age). Ageing resulted in the increase in receptor for advanced glycation endproducts expression, as well as the entrance of thrombin and albumin in hippocampal parenchyma. In contrast, the loss of platelet-derived growth factor receptor-β (PDGFR-β) positive cells, in both regions, was only related to AD pathogenesis. Hypovascularization was affected by both ageing and AD in the hippocampus, but resulted from the interaction between both factors in the cortex. Astrogliosis was a result of AD in hippocampus and of both factors in cortex, while microgliosis was associated with fibrillar amyloid plaques in AD-like mice and with the interaction between both factors in each of the studied regions. In sum, these data show that senile plaques precede vascular and glial alterations only in hippocampus, whereas in cortex, vascular and glial alterations, namely the loss of PDGFR-β-positive cells and astrogliosis, accompanied the first senile plaques. Hence, this study points to vascular and glial events that co-exist in AD pathogenesis and age-related brain vulnerabilities.

Bilirubin-induced neurologic dysfunction (BIND) and classical kernicterus are clinical manifestations of moderate to severe hyperbilirubinemia whenever bilirubin levels exceed the capacity of the brain defensive mechanisms in preventing its entrance and cytotoxicity. In such circumstances and depending on the associated co-morbidities, bilirubin accumulation may lead to short- or long-term neurodevelopmental disabilities, which may include deficits in auditory, cognitive, and motor processing. Neuronal cell death, astrocytic reactivity, and microglia activation are part of the bilirubin-induced pathogenesis. Less understood is how abnormal growth and maturation of oligodendrocytes may impact on brain development, affecting the formation of myelin tracts. Based on in-vitro and in-vivo models, as well as in clinical cases presented here, we propose the existence of impaired myelination by bilirubin with long-term sequelae, mainly in pre-term infants. Sensitive time-windows are highlighted and centered on the different developmental-dependent impairments determined by bilirubin, and the influence of sepsis and hypoxia is reviewed.

Alzheimer’s disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive Aβ production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM Aβ1–42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after Aβ treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated β-galactosidase (SA-β-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by Aβ in 2 DIV microglia. Additionally, Aβ downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aβ treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder affecting motor neurons (MNs). Evidences indicate that ALS is a non-cell autonomous disease in which glial cells participate in both disease onset and progression. Exosomal transfer of mutant copper-zinc superoxide dismutase 1 (mSOD1) from cell-to-cell was suggested to contribute to disease dissemination. Data from our group and others showed that exosomes from activated cells contain inflammatory-related microRNAs (inflamma-miRNAs) that recapitulate the donor cell. While glia-derived exosomes and their effects in neurons have been addressed by several studies, only a few investigated the influence of motor neuron (MN)-derived exosomes in other cell function, the aim of the present study. We assessed a set of inflamma-miRs in NSC-34 MN-like cells transfected with mutant SOD1(G93A) and extended the study into their derived exosomes (mSOD1 exosomes). Then, the effects produced by mSOD1 exosomes in the activation and polarization of the recipient N9 microglial cells were investigated. Exosomes in coculture with N9 microglia and NSC-34 cells [either transfected with either wild-type (wt) human SOD1 or mutant SOD1(G93A)] showed to be transferred into N9 cells. Increased miR-124 expression was found in mSOD1 NSC-34 cells and in their derived exosomes. Incubation of mSOD1 exosomes with N9 cells determined a sustained 50% reduction in the cell phagocytic ability. It also caused a persistent NF-kB activation and an acute generation of NO, MMP-2, and MMP-9 activation, as well as upregulation of IL-1β, TNF-α, MHC-II, and iNOS gene expression, suggestive of induced M1 polarization. Marked elevation of IL-10, Arginase 1, TREM2, RAGE, and TLR4 mRNA levels, together with increased miR-124, miR-146a, and miR-155, at 24 h incubation, suggest the switch to mixed M1 and M2 subpopulations in the exosome-treated N9 microglial cells. Exosomes from mSOD1 NSC-34 MNs also enhanced the number of senescent-like positive N9 cells. Data suggest that miR-124 is translocated from the mSOD1 MNs to exosomes, which determine early and late phenotypic alterations in the recipient N9-microglial cells. In conclusion, modulation of the inflammatory-associated miR-124, in mSOD1 NSC-34 MNs, with potential benefits in the cargo of their exosomes may reveal a promising therapeutic strategy in halting microglia activation and associated effects in MN degeneration.

The blood–brain barrier (BBB) is a complex and dynamic structure that plays a key role in central nervous system (CNS) homeostasis. It strictly regulates the entrance of molecules into the brain parenchyma and prevents the access of neurotoxins and pathogens while promoting the efflux of several molecules. The brain microvascular endothelial cells are the anatomical basis of the BBB, which has unique characteristics such as the elaborate junctional complexes that nearly obliterate the intercellular space as well as the presence of influx and efflux transporters. Endothelial cells establish important interactions with glial cells, neurons, and perivascular pericytes as well as with the acellular components of the basement membrane, which together constitute the neurovascular unit. BBB disruption has been reported in a wide range of CNS pathologies, with an emerging role in the onset and disease progression. Accordingly, recent studies revealed vascular dysfunction in neonatal jaundice, a common pathology in the early neonatal period affecting 1/10 children presenting values of total bilirubin >17 mg/dL (291 μM). Here we summarize the clinical aspects of moderate to severe neonatal jaundice and provide a comprehensive review of the literature regarding bilirubin-induced neurotoxicity from a vascular-centered approach. The collected evidence place endothelial dysfunction and pericyte demise as key players in the disruption of CNS homeostasis, mainly in cases of lasting hyperbilirubinemia, thus pointing to novel targets to prevent neurological dysfunction due to severe neonatal jaundice.

There is an unmet clinical need to develop neuroprotective agents for cerebrovascular procedures requiring transient cerebral artery occlusion. This study aims to investigate the effects of a single pre-treatment dose of 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a glycogen synthase kinase-3β (GSK-3β) inhibitor, in a transient focal cerebral ischemia model. Twenty-eight male adult Wistar rats were subjected to right middle cerebral artery (MCA) occlusion via intraluminal thread technique for 60 min under continuously cortical perfusion monitoring by laser-Doppler flowmetry. Rats were divided into two groups: control or treatment groups. In the treated group, TDZD-8 (5 mg/kg; intravenously) was administered 10 min before the onset of the MCA ischemia. At 24-h reperfusion, the following parameters were evaluated: neurological deficits, brain infarct volume, ipsilateral hemispheric oedema, neuron specific enolase (NSE) plasma levels, parenchyma histology (H–E staining), Fluoro-Jade positive neurons, p-Akt and total Akt expression by western blot analysis and p-Akt-positive nuclei by immunohistochemistry. Infarct volume (P

Neural stem cells (NSC) are self-renewing multipotent cells that have emerged as a powerful tool to repair the injured brain. These cells can be cultured as neurospheres, which are floating aggregates of neural stem/progenitor cells (NSPCs). Despite their high clonal expansion capacity, it has been suggested that in neurospheres, only a small percentage of cells are capable of proliferation and that this system is not efficient in terms of neurogenic competence. Thus, our aim was to develop a neurosphere culture method with a highly proliferative stem/progenitor cell population and particularly with a prominent neurogenic potential, surpassing some of the claimed weaknesses of the neurosphere assay. In our model, mouse neurospheres were harvested from neural tissue at E15 and after only 4 days in vitro (DIV), we have achieved highly proliferative primary neurospheres (81% Sox2 and 76% Ki67 positive cells) and a rather low number of cells expressing glial and neuronal markers (∼10%). After inducing differentiation, we have attained an enriched neuronal population (45% β-III-tubulin positive cells at 15 DIV). Using a simple methodology, we have developed a NSPC model that can provide a valuable source of neuronal precursors, thus offering a potential starting point for cell replacement therapies following CNS injury.

Object There is an unmet clinical need to develop neuroprotective agents for neurosurgical and endovascular procedures that require transient cerebral artery occlusion. The aim in this study was to explore the effects of a single dose of recombinant human erythropoietin (rhEPO) before middle cerebral artery (MCA) occlusion in a focal cerebral ischemia/reperfusion model. Methods Twenty-eight adult male Wistar rats were subjected to right MCA occlusion via the intraluminal thread technique for 60 minutes under continuous cortical perfusion monitoring by laser Doppler flowmetry. Rats were divided into 2 groups: control and treatment. In the treated group, rhEPO (1000 IU/kg intravenously) was administered 10 minutes before the onset of the MCA ischemia. At 24-hour reperfusion, animals were examined for neurological deficits, blood samples were collected, and animals were killed. The following parameters were evaluated: brain infarct volume, ipsilateral hemispheric edema, neuron-specific enolase plasma levels, parenchyma histological features (H & E staining), Fluoro-Jade–positive neurons, p-Akt and total Akt expression by Western blot analysis, and p-Akt–positive nuclei by immunohistochemical investigation. Results Infarct volume and Fluoro-Jade staining of degenerating neurons in the infarct area did not vary between groups. The severity of neurological deficit (p < 0.001), amount of brain edema (78% reduction in treatment group, p < 0.001), and neuron-specific enolase plasma levels (p < 0.001) were reduced in the treatment group. Perivascular edema was histologically less marked in the treatment group. No variations in the expression or localization of p-Akt were seen. Conclusions Administration of rhEPO before the onset of 60-minute transient MCA ischemia protected the brain from this insult. It is unlikely that rhEPO pretreatment leads to direct neuronal antiapoptotic effects, as supported by the lack of Akt activation, and its benefits are most probably related to an indirect effect on brain edema as a consequence of blood-brain barrier preservation. Although research on EPO derivatives is increasing, rhEPO acts through distinct neuroprotective pathways and its clinical safety profile is well known. Clinically available rhEPO is a potential therapy for prevention of neuronal injury induced by transitory artery occlusion during neurovascular procedures.

Background Kernicterus is an irreversible brain damage caused by bilirubin deposition in selective brain regions. Sick and preterm infants with hyperbilirubinemia are particularly susceptible to the condition. Methods We studied autopsied brain tissue from a premature female infant with kernicterus with a bilirubin:albumin molar ratio of 1.0, hypoxia, acidosis, and seizures. The patient, previously described as having cerebellar axon/myelin loss and angiogenic sprouting, was assessed for histopathological features in brain regions less investigated, such as hippocampus and corpus striatum. Results were compared with age-matched controls. Results Increased blood vessel density with poorly defined lumen structures was observed in the mesencephalon, pons, and medulla oblongata, and, more predominantly, in the corpus striatum and hippocampus. These two regions exhibited increased expression of vascular endothelial growth factor, paralleled by vascular endothelial growth factor receptor-2, and albumin extravasation into the brain parenchyma. No similar findings were observed in the nonjaundiced babies with hypoxia that served as controls (one preterm with sepsis and a term infant with pneumonia). We found increased cellular expression of multidrug resistance–associated protein 1 and P-glycoprotein in the hippocampus, known as defensive mechanisms against bilirubin-induced cytotoxicity. Increased density of blood vessels and microvascular permeability, together with parenchymal albumin, may have contributed to increasing the brain content and retention of bilirubin, a condition implicated in kernicterus disease. Conclusions This novel finding in a premature baby with kernicterus and associated risk factors deserves to be investigated in similar patients to better understand the less-well described effects of bilirubin-induced neurological sequelae in preterm infants.

Glioblastoma (GBM) is the most common and aggressive type of brain tumor in adults. The triazene Temozolomide (TMZ), an alkylating drug, is the classical chemotherapeutic agent for gliomas, but has been disappointing against the highly invasive and resistant nature of GBM. Hybrid compounds may open new horizons within this challenge. The multicomponent therapeutic strategy here used resides on a combination of two repurposing drugs acting by different but potentially synergistic mechanisms, improved efficacy, and lower resistance effects. We synthesized a new hybrid compound (HYBCOM) by covalently binding a TMZ analogue to valproic acid, a histone deacetylase inhibitor drug that was shown to sensitize TMZ-resistant glioma cells. Advantages of this new molecule as compared to TMZ, in terms of chemotherapeutic efficacy, were investigated. Our results evidenced that HYBCOM more efficiently decreased the viability and proliferation of the GL261 glioma cells, while showing to better target the tumor cells than the functionally normal astrocytes. Increased cytotoxicity by HYBCOM may be a consequence of the improved autophagic process observed. Additionally, HYBCOM changed the morphology of GL261 cells into a nonpolar, more rounded shape, impairing cell migration ability. Most interesting, and in opposite to TMZ, cells exposed to HYBCOM did not enhance the expression of drug resistance proteins, a major issue in the treatment of GBM. Overall, our studies indicate that HYBCOM has promising chemotherapeutic benefits over the classical TMZ, and future studies should assess if the treatment translates into efficacy in glioblastoma experimental models and reveal clinical benefits in GBM patients.

Natural food sources constitute a promising source of new compounds with neuroprotective properties, once they have the ability to reach the brain. Our aim was to evaluate the brain accessibility of quercetin, epigallocatechin gallate (EGCG) and cyanidin-3-glucoside (C3G) in relation to their neuroprotective capability. Primary cortical neuron cultures were exposed to oxidative insult in the absence and presence of the selected compounds, and neuroprotection was assessed through evaluation of apoptotic-like and necrotic-like cell death. The brain accessibility of selected compounds was assessed using an optimised human blood-brain barrier model. The blood-brain barrier model was crossed rapidly by EGCG and more slowly by C3G, but not by quercetin. EGCG protected against oxidation-induced neuronal necrotic-like cell death by ~40%, and apoptosis by ~30%. Both quercetin and C3G were less effective, since only the lowest quercetin concentration was protective, and C3G only prevented necrosis by ~37%. Quercetin, EGCG and C3G effectively inhibited α-synuclein fibrillation over the relevant timescale applied here. Overall, EGCG seems to be the most promising neuroprotective compound. Thus, inclusion of this polyphenol in the diet might provide an affordable means to reduce the impact of neurodegenerative diseases.

Research on the mechanisms of bilirubin-induced neurological dysfunction focuses mainly on neuronal death, astrocyte-mediated events and microglia activation. Although myelin damage by unconjugated bilirubin (UCB) has been documented in neonatal kernicterus cases, the events leading to myelination impairment were never explored. This condition may occur by reduced oligodendrocyte precursor cells (OPC) number, or failure of OPC to differentiate in myelinating oligodendrocytes. We have shown that UCB elicits an inflammatory response, glutamate release and reactive oxygen species (ROS) generation in neurons and glial cells, biomolecules with toxic properties on OPC. Hence, we propose to examine whether UCB determines OPC demise and, if so, which signaling pathways are involved. Our results show that OPC display increased apoptosis and necrosis-like cell death upon UCB exposure, mediated by early signals of endoplasmic reticulum (ER) stress [e.g. upregulation of glucose-regulated protein (GRP)78, inositol-requiring enzyme (IRE)-1α and activation transcription factor (ATF)-6, as well as activation of caspase-2 and c-Jun N-terminal kinase (JNK)], followed by mitochondrial dysfunction (e.g. loss of mitochondria membrane potential and caspase-9 activation). The later calpain activation points to intracellular Ca(2+) overload and intervention of both ER and mitochondria. Downstream production of ROS may derive from mitochondria damage and secondary injuries, possibly determining the second cycle of GRP78, IRE-1α, caspase-2 and JNK activation. Moreover, inhibition of caspases, calpains and oxidative stress, by using specific inhibitors, prevented UCB-induced OPC death. UCB did not induce the release of cytokines or glutamate by OPC. These results indicate that UCB by reducing OPC survival, through a cascade of programmed intracellular events triggered by ER stress and mitochondria dysfunction, can compromise myelinogenesis.

Tight junctions are elaborate networks of transmembrane and cytosolic proteins that regulate epithelial permeability. Tricellulin was the first tight junction protein found at tricellular tight junctions, the specialized structures occurring where three cells meet together. Here, we summarize the current knowledge about tricellulin (marvelD2), a MARVEL domain protein. We address tricellulin location at tricellular junctions, and establish the comparison with the other members of the MARVEL family, occludin (marvelD1) and marvelD3. The structure of tricellulin and its membrane folding, as well as the proposed molecular interactions of tricellulin with other tight junction proteins, together with the interplay between those proteins are also discussed. In addition, we address the role of tricellulin in barrier properties, discriminating the involvement of the protein in paracellular permeability at bicellular and at tricellular tight junctions. Moreover, the key importance of the protein for hearing is highlighted based on the fact that mutations in TRIC, the human tricellulin gene, lead to deafness. Furthermore, this review points to some of the aspects that still deserve clarification for a better understanding of the biology of tight junctions in general and of tricellulin in particular.

Hippocampus is one of the brain regions most vulnerable to unconjugated bilirubin (UCB) encephalopathy, although cerebellum also shows selective yellow staining in kernicterus. We previously demonstrated that UCB induces oxidative stress in cortical neurons, disruption of neuronal network dynamics, either in developing cortical or hippocampal neurons, and that immature cortical neurons are more prone to UCB-induced injury. Here, we studied if immature rat neurons isolated from cortex, cerebellum and hippocampus present distinct features of oxidative stress and cell dysfunction upon UCB exposure. We also explored whether oxidative damage and its regulation contribute to neuronal dysfunction induced by hyperbilirubinemia, considering neurite extension and ramification, as well as cell death. Our results show that UCB induces nitric oxide synthase expression, as well as production of nitrites and cyclic guanosine monophosphate in immature neurons, mainly in those from hippocampus. After exposure to UCB, hippocampal neurons presented the highest content of reactive oxygen species, disruption of glutathione redox status and cell death, when compared to neurons from cortex or cerebellum. In particular, the results indicate that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein implicated in the maintenance of cellular oxidation status. However, longer neuronal exposure to UCB caused down-regulation of DJ-1 expression, especially in hippocampal neurons. In addition, a greater impairment in neurite outgrowth and branching following UCB treatment was also noticed in immature neurons from hippocampus. Interestingly, pre-incubation with N-acetylcysteine, a precursor of glutathione synthesis, protected neurons from UCB-induced oxidative stress and necrotic cell death, preventing DJ-1 down-regulation and neuritic impairment. Taken together, these data point to oxidative injury and disruption of neuritic network as hallmarks in hippocampal susceptibility to UCB. Most importantly, they also suggest that local differences in glutathione content may account to the different susceptibility between brain regions exposed to UCB.

Encephalopathy by hyperbilirubinemia in infants has been described for decades, but neither the underlying cellular and molecular mechanisms nor the selective pattern of bilirubin deposition in the brain is well understood. The brain is composed of highly specialized and diverse populations of cells, represented by neurons and glia that comprise astrocytes, oligodendrocytes, and microglia. Although microscopic evaluation of icteric brain sections revealed bilirubin within neurons, neuronal processes, and microglia, cell dependent-sensitivity to bilirubin toxicity and the role of each nerve cell type are poorly understood. Even less considered are glial and neuronal pathologic alterations as integrated phenomena. The available knowledge on reactivity of glial cells to bilirubin and on the impairment to neuronal network dynamics that it causes, here summarized, suggests that a better comprehension of the interplay between neurons and glia is essential to understand bilirubin neurotoxicity and highlight potential molecular targets that may lead to disease-modifying therapeutic approaches.

Hyperbilirubinemia may lead to encephalopathy in neonatal life, particularly in premature infants. Although the mechanisms were never established, clinicians commonly consider sepsis as a risk factor for bilirubin-induced neurological dysfunction (BIND). Our previous studies showed that elevated levels of unconjugated bilirubin (UCB) have immunostimulant effects, which are potentiated by lipopolysaccharide (LPS), and that immature neural cells are more vulnerable to UCB. The present study was undertaken to explore the role of nitric oxide (NO)/NO synthase (NOS), c-Jun N-terminal kinases (JNK) 1/2 and caspase activation in BIND, as well as the additional effects of inflammation, in immature neurons, incubated from 1 h to 24 h, at 37°C. UCB, at conditions mimicking those of jaundiced newborns (UCB/serum albumin=0.5), induced NO production, neuronal NOS (nNOS) expression and JNK1/2 activation in 3 days in vitro neuron cultures. As a consequence of these events, mitochondrial and extrinsic pathways of apoptosis were initiated, ultimately leading to neuronal dysfunction. Co-incubation with TNF-α+IL-1β intensified the activation of NO/NOS, JNK1/2, caspase-8, caspase-9 and caspase-3 by UCB. Cleavage of Bid into truncated Bid (tBid), as well as increased cytotoxic potential, were also observed. Interestingly, both L-NAME (NOS inhibitor) and SP600125 (JNK1/2 inhibitor) reversed the effects produced by UCB either alone, or in association with pro-inflammatory cytokines. Taken together, our data reveal not only that activation of NO/NOS, JNK1/2 and caspase cascades are important determinants of BIND, but also that the association of TNF-α+IL-1β have cumulative effects. These events provide a reason for the risk of sepsis in BIND and point to potential targets for therapeutic intervention.