The Evolving Landscape of Neurotoxicity by Unconjugated Bilirubin: Role of Glial Cells and Inflammation

Unconjugated hyperbilirubinemia is a common condition in the first week of postnatal life. Although generally harmless, some neonates may develop very high levels of unconjugated bilirubin (UCB), which may surpass the protective mechanisms of the brain in preventing UCB accumulation. In this case, both short-term and long-term neurodevelopmental disabilities, such as acute and chronic UCB encephalopathy, known as kernicterus, or more subtle alterations defined as bilirubin-induced neurological dysfunction (BIND) may be produced. There is a tremendous variability in babies’ vulnerability toward UCB for reasons not yet explained, but preterm birth, sepsis, hypoxia, and hemolytic disease are comprised as risk factors. Therefore, UCB levels and neurological abnormalities are not strictly correlated. Even nowadays, the mechanisms of UCB neurotoxicity are still unclear, as are specific biomarkers, and little is known about lasting sequelae attributable to hyperbilirubinemia. On autopsy, UCB was shown to be within neurons, neuronal processes, and microglia, and to produce loss of neurons, demyelination, and gliosis. In isolated cell cultures, UCB was shown to impair neuronal arborization and to induce the release of pro-inflammatory cytokines from microglia and astrocytes. However, cell dependent sensitivity to UCB toxicity and the role of each nerve cell type remains not fully understood. This review provides a comprehensive insight into cell susceptibilities and molecular targets of UCB in neurons, astrocytes, and oligodendrocytes, and on phenotypic and functional responses of microglia to UCB. Interplay among glia elements and cross-talk with neurons, with a special emphasis in the UCB-induced immunostimulation, and the role of sepsis in BIND pathogenesis are highlighted. New and interesting data on the anti-inflammatory and antioxidant activities of different pharmacological agents are also presented, as novel and promising additional therapeutic approaches to BIND

Synaptic Failure: Focus in an Integrative View of ALS

From early description by Charcot, the classification of the Amyotrophic Lateral Sclerosis (ALS) is evolving from a subtype of Motor Neuron (MN) Disease to be considered rather a multi-systemic, non-cell autonomous and complex neurodegenerative disease. In the last decade, the huge amount of knowledge acquired has shed new insights on the pathological mechanisms underlying ALS from different perspectives. However, a whole vision on the multiple dysfunctional pathways is needed with the inclusion of information often excluded in other published revisions. We propose an integrative view of ALS pathology, although centered on the synaptic failure as a converging and crucial player to the etiology of the disease. Homeostasis of input and output synaptic activity of MNs has been proved to be severely and early disrupted and to definitively contribute to microcircuitry alterations at the spinal cord. Several cells play roles in synaptic communication across the MNs network system such as interneurons, astrocytes, microglia, Schwann and skeletal muscle cells. Microglia are described as highly dynamic surveying cells of the nervous system but also as determinant contributors to the synaptic plasticity linked to neuronal activity. Several signaling axis such as TNFα/TNFR1 and CX3CR1/CX3CL1 that characterize MN-microglia cross talk contribute to synaptic scaling and maintenance, have been found altered in ALS. The presence of dystrophic and atypical microglia in late stages of ALS, with a decline in their dynamic motility and phagocytic ability, together with less synaptic and neuronal contacts disrupts the MN-microglia dialogue, decreases homeostatic regulation of neuronal activity, perturbs “on/off” signals and accelerates disease progression associated to impaired synaptic function and regeneration. Other hotspot in the ALS affected network system is the unstable neuromuscular junction (NMJ) leading to distal axonal degeneration. Reduced neuromuscular spontaneous synaptic activity in ALS mice models was also suggested to account for the selective vulnerability of MNs and decreased regenerative capability. Synaptic destabilization may as well derive from increased release of molecules by muscle cells (e.g. NogoA) and by terminal Schwann cells (e.g. semaphorin 3A) conceivably causing nerve terminal retraction and denervation, as well as inhibition of re-connection to muscle fibers. Indeed, we have overviewed the alterations on the metabolic pathways and self-regenerative capacity presented in skeletal muscle cells that contribute to muscle wasting in ALS. Finally, a detailed footpath of pathologic changes on MNs and associated dysfunctional and synaptic alterations is provided. The oriented motivation in future ALS studies as outlined in the present article will help in fruitful novel achievements on the mechanisms involved and in developing more target-driven therapies that will bring new hope in halting or delaying disease progression in ALS patients

Microglia Susceptibility to Free Bilirubin Is Age-Dependent

Funding: This work was supported by National Funds (Fundacão para a Ciencia e a Tecnologia—UID/DTP/04138/2015-2019) to iMed.ULisboa. AF had a post-doctoral research position (C2007-FFUL/UBMBE/02/ 2011) and AV a post-doc fellowship (SFRH/BPD/76590/2011), both granted by FCT. CS and ES were Master students from University of Bologna who developed their thesis at Universidade de Lisboa, with fellowships from Erasmus+ Programme. We thank Professor Stefano Girotti from the University of Bologna for establishing such collaborative Program and for the local supervision of the students. The funding organization had no role in data collection and analysis, decision to publish, or preparation of the manuscripIncreased concentrations of unconjugated bilirubin (UCB), namely its free fraction (Bf), in neonatal life may cause transient or definitive injury to neurons and glial cells. We demonstrated that UCB damages neurons and glial cells by compromising oligodendrocyte maturation and myelination, and by activating astrocytes and microglia. Immature neurons and astrocytes showed to be especially vulnerable. However, whether microglia susceptibility to UCB is also age-related was never investigated. We developed a microglia culture model in which cells at 2 days in vitro (2DIV) revealed to behave as the neonatal microglia (amoeboid/reactive cells), in contrast with those at 16DIV microglia that performed as aged cells (irresponsive/dormant cells). Here, we aimed to unveil whether UCB-induced toxicity diverged from the young to the long-cultured microglia. Cells were isolated from the cortical brain of 1- to 2-day-old CD1 mice and incubated for 24 h with 50/100 nM Bf levels, which were associated to moderate and severe neonatal hyperbilirubinemia, respectively. These concentrations of Bf induced early apoptosis and amoeboid shape in 2DIV microglia, while caused late apoptosis in 16DIV cells, without altering their morphology. CD11b staining increased in both, but more markedly in 2DIV cells. Likewise, the gene expression of HMGB1, a well-known alarmin, as well as HMGB1 and GLT-1–positive cells, were enhanced as compared to long-maturated microglia. The CX3CR1 reduction in 2DIV microglia was opposed to the 16DIV cells and suggests a preferential Bf-induced sickness response in younger cells. In conformity, increased mitochondrial mass and NO were enhanced in 2DIV cells, but unchanged or reduced, respectively, in the 16DIV microglia. However, 100 nM Bf caused iNOS gene overexpression in 2DIV and 16DIV cells. While only arginase 1/IL-1β gene expression levels increased upon 50/100 nM Bf treatment in long-maturated microglia, MHCII/arginase 1/TNF-α/IL-1β/IL-6 (>10-fold) were upregulated in the 2DIV microglia. Remarkably, enhanced inflammatory-associated microRNAs (miR-155/miR-125b/miR-21/miR-146a) and reduced anti-inflammatory miR-124 were found in young microglia by both Bf concentrations, while remained unchanged (miR/21/miR-125b) or decreased (miR-155/miR-146a/miR-124) in aged cells. Altogether, these findings support the neurodevelopmental susceptibilities to UCB-induced neurotoxicity, the most severe disabilities in premature babies, and the involvement of immune-inflammation neonatal microglia processes in poorer outcomes.publishersversionpublishe

Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

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

Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

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

Phenotypic Effects of Wild-Type and Mutant SOD1 Expression in N9 Murine Microglia at Steady State, Inflammatory and Immunomodulatory Conditions

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

Intrathecal injection of the secretome from ALS motor neurons regulated for miR-124 expression prevents disease outcomes in SOD1-G93A mice

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)

S100B inhibition protects from chronic experimental autoimmune encephalomyelitis

© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.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.This work was funded by grants from Ordem dos Farmacêuticos (AF), Merck KGaA (AF) and Fundação de Ciênciase Tecnologia (UIDB/04138/2020; UIDP/04138/2020) (iMed.ULisboa).info:eu-repo/semantics/publishedVersio

Recovery of depleted miR-146a in ALS cortical astrocytes reverts cell aberrancies and prevents paracrine pathogenicity on microglia and motor neurons

Copyright © 2021 Barbosa, Gomes, Sequeira, Gonçalves-Ribeiro, Pina, Carvalho, Moreira, Vaz, Vaz and Brites. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).Reactive astrocytes in Amyotrophic Lateral Sclerosis (ALS) change their molecular expression pattern and release toxic factors that contribute to neurodegeneration and microglial activation. We and others identified a dysregulated inflammatory miRNA profile in ALS patients and in mice models suggesting that they represent potential targets for therapeutic intervention. Such cellular miRNAs are known to be released into the secretome and to be carried by small extracellular vesicles (sEVs), which may be harmful to recipient cells. Thus, ALS astrocyte secretome may disrupt cell homeostasis and impact on ALS pathogenesis. Previously, we identified a specific aberrant signature in the cortical brain of symptomatic SOD1-G93A (mSOD1) mice, as well as in astrocytes isolated from the same region of 7-day-old mSOD1 mice, with upregulated S100B/HMGB1/Cx43/vimentin and downregulated GFAP. The presence of downregulated miR-146a on both cases suggests that it can be a promising target for modulation in ALS. Here, we upregulated miR-146a with pre-miR-146a, and tested glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS) for their immunoregulatory properties. VS was more effective in restoring astrocytic miR-146a, GFAP, S100B, HMGB1, Cx43, and vimentin levels than GUDCA, which only recovered Cx43 and vimentin mRNA. The miR-146a inhibitor generated typical ALS aberrancies in wild type astrocytes that were abolished by VS. Similarly, pre-miR-146a transfection into the mSOD1 astrocytes abrogated aberrant markers and intracellular Ca2+ overload. Such treatment counteracted miR-146a depletion in sEVs and led to secretome-mediated miR-146a enhancement in NSC-34-motor neurons (MNs) and N9-microglia. Secretome from mSOD1 astrocytes increased early/late apoptosis and FGFR3 mRNA in MNs and microglia, but not when derived from pre-miR-146a or VS-treated cells. These last strategies prevented the impairment of axonal transport and synaptic dynamics by the pathological secretome, while also averted microglia activation through either secretome, or their isolated sEVs. Proteomic analysis of the target cells indicated that pre-miR-146a regulates mitochondria and inflammation via paracrine signaling. We demonstrate that replenishment of miR-146a in mSOD1 cortical astrocytes with pre-miR-146a or by VS abrogates their phenotypic aberrancies and paracrine deleterious consequences to MNs and microglia. These results propose miR-146a as a new causal and emerging therapeutic target for astrocyte pathogenic processes in ALS.This work was supported by the Research Grant of the Santa Casa Scientific Research Program on ALS, by Santa Casa da Misericórdia de Lisboa (SCML), Portugal, Project Ref. ELA-2015-002 (to DB). Fundação para a Ciência e a Tecnologia (FCT) also supported the project PTDC/MED-NEU/31395/2017 (to DB), PTDC/MED-QUI/30021/2017 (to RM) and iMed. ULisboa (UIDB/04138/2020 and UIDP/04138/2020), together with Programa Operacional Regional de Lisboa and the Programa Operacional Competitividade e Internacionalização (LISBOA-01-0145-FEDER-031395 to DB). Individual fellowships MB (SFRH/BD/129586/2017), CG (SFRH/BD/102718/2014), AV (SFRH/BPD/76590/2011), and JG-R PD/BD/150342/2019 were from FCT. CS was a research fellowship recipient from SCML.info:eu-repo/semantics/publishedVersio