Role of copper in central nervous system physiology and pathology.

Copper is a transition metal and an essential element for the organism, as alterations in its homeostasis leading to metal accumulation or deficiency have pathological effects in several organs, including the central nervous system. Central copper dysregulations have been evidenced in two genetic disorders characterized by mutations in the copper-ATPases ATP7A and ATP7B, Menkes disease and Wilson's disease, respectively, and also in multifactorial neurological disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis. This review summarizes current knowledge about the role of copper in central nervous system physiology and pathology, reports about unbalances in copper levels and/or distribution under disease, describes relevant animal models for human disorders where copper metabolism genes are dysregulated, and discusses relevant therapeutic approaches modulating copper availability. Overall, alterations in copper metabolism may contribute to the etiology of central nervous system disorders and represent relevant therapeutic targets to restore tissue homeostasis.

Publicly available ex vivo transcriptomics datasets to explore CNS physiology and neurodegeneration: state of the art and perspectives.

The central nervous system (CNS) is characterized by an intricate composition of diverse cell types, including neurons and glia cells (astrocytes, oligodendrocytes, and microglia), whose functions may differ along time, between sexes and upon pathology. The advancements in high-throughput transcriptomics are providing fundamental insights on cell phenotypes, so that molecular codes and instructions are ever more described for CNS physiology and neurodegeneration. To facilitate the search of relevant information, this review provides an overview of key CNS transcriptomics studies ranging from CNS development to ageing and from physiology to pathology as defined for five neurodegenerative disorders and their relative animal models, with a focus on molecular descriptions whose raw data were publicly available. Accurate phenotypic descriptions of cellular states correlate with functional changes and this knowledge may support research devoted to the development of therapeutic strategies supporting CNS repair and function.

iAstrocytes do not restrain T cell proliferation in vitro.

The cross-talk between T cells and astrocytes occurring under physiological and, even more, neuroinflammatory conditions may profoundly impact the generation of adaptive immune responses in the nervous tissue. In this study, we used a standardized in vitro co-culture assay to investigate the immunomodulatory properties of astrocytes differing for age, sex, and species. Mouse neonatal astrocytes enhanced T cell vitality but suppressed T lymphocyte proliferation in response to mitogenic stimuli or myelin antigens, regardless of the Th1, Th2 or Th17 T cell phenotype. Studies comparing glia cells from adult and neonatal animals showed that adult astrocytes were more efficient in inhibiting T lymphocyte activation than neonatal astrocytes, regardless of their sex. Differently from primary cultures, mouse and human astrocytes derived from reprogrammed fibroblasts did not interfere with T cell proliferation. Overall, we describe a standardized astrocyte-T cell interaction in vitro assay and demonstrate that primary astrocytes and iAstrocytes may differ in modulating T cell function.

HNF4α, SP1 and c-myc are master regulators of CNS autoimmunity.

Hepatocyte nuclear factor 4 α (HNF4α), a transcription factor (TF) essential for embryonic development, has been recently shown to regulate the expression of inflammatory genes. To characterize HNF4a function in immunity, we measured the effect of HNF4α antagonists on immune cell responses in vitro and in vivo. HNF4α blockade reduced immune activation in vitro and disease severity in the experimental model of multiple sclerosis (MS). Network biology studies of human immune transcriptomes unraveled HNF4α together with SP1 and c-myc as master TF regulating differential expression at all MS stages. TF expression was boosted by immune cell activation, regulated by environmental MS risk factors and higher in MS immune cells compared to controls. Administration of compounds targeting TF expression or function demonstrated non-synergic, interdependent transcriptional control of CNS autoimmunity in vitro and in vivo. Collectively, we identified a coregulatory transcriptional network sustaining neuroinflammation and representing an attractive therapeutic target for MS and other inflammatory disorders.

Astrocytes and Microglia in Stress-Induced Neuroinflammation: The African Perspective.

Background: Africa is laden with a youthful population, vast mineral resources and rich fauna. However, decades of unfortunate historical, sociocultural and leadership challenges make the continent a hotspot for poverty, indoor and outdoor pollutants with attendant stress factors such as violence, malnutrition, infectious outbreaks and psychological perturbations. The burden of these stressors initiate neuroinflammatory responses but the pattern and mechanisms of glial activation in these scenarios are yet to be properly elucidated. Africa is therefore most vulnerable to neurological stressors when placed against a backdrop of demographics that favor explosive childbearing, a vast population of unemployed youths making up a projected 42% of global youth population by 2030, repressive sociocultural policies towards women, poor access to healthcare, malnutrition, rapid urbanization, climate change and pollution. Early life stress, whether physical or psychological, induces neuroinflammatory response in developing nervous system and consequently leads to the emergence of mental health problems during adulthood. Brain inflammatory response is driven largely by inflammatory mediators released by glial cells; namely astrocytes and microglia. These inflammatory mediators alter the developmental trajectory of fetal and neonatal brain and results in long-lasting maladaptive behaviors and cognitive deficits. This review seeks to highlight the patterns and mechanisms of stressors such as poverty, developmental stress, environmental pollutions as well as malnutrition stress on astrocytes and microglia in neuroinflammation within the African context.

Lessons from S1P receptor targeting in multiple sclerosis.

Sphingosine 1-phosphate (S1P) is a potent bioactive sphingolipid binding to specific G protein-coupled receptors expressed in several organs. The relevance of S1P-S1P receptor axis in the pathophysiology of immune and nervous systems has encouraged the development of S1P receptor modulators for the treatment of neurological, autoimmune and/or inflammatory disorders. Currently, four S1P receptor modulators are approved drugs for multiple sclerosis (MS), an inflammatory disorder of the central nervous system. As main pharmacologic effect, these treatments induce lymphopenia due to the loss of responsiveness to S1P gradients guiding lymphocyte egress from lymphoid organs into the bloodstream. Recent data point to immunological effects of the S1P modulators beyond the inhibition of lymphocyte trafficking. Further, these drugs may cross the blood-brain barrier and directly target CNS resident cells expressing S1P receptors. Here we review the role of S1P signalling in neuroimmunology at the light of the evidences generated from the study of the mechanism of action of S1P receptor modulators in MS and integrate this information with findings derived from neuroinflammatory animal models and in vitro observations. These insights can direct the application of therapeutic approaches targeting S1P receptors in other disease areas.

JAB1 deletion in oligodendrocytes causes senescence-induced inflammation and neurodegeneration in mice.

Oligodendrocytes are the primary target of demyelinating disorders, and progressive neurodegenerative changes may evolve in the CNS. DNA damage and oxidative stress are considered key pathogenic events, but the underlying molecular mechanisms remain unclear. Moreover, animal models do not fully recapitulate human diseases, complicating the path to effective treatments. Here we report that mice with cell-autonomous deletion of the nuclear COP9 signalosome component CSN5 (JAB1) in oligodendrocytes develop DNA damage and defective DNA repair in myelinating glial cells. Interestingly, oligodendrocytes lacking JAB1 expression underwent a senescence-like phenotype that fostered chronic inflammation and oxidative stress. These mutants developed progressive CNS demyelination, microglia inflammation, and neurodegeneration, with severe motor deficits and premature death. Notably, blocking microglia inflammation did not prevent neurodegeneration, whereas the deletion of p21CIP1 but not p16INK4a pathway ameliorated the disease. We suggest that senescence is key to sustaining neurodegeneration in demyelinating disorders and may be considered a potential therapeutic target.

Dysregulated copper transport in multiple sclerosis may cause demyelination via astrocytes.

Demyelination is a key pathogenic feature of multiple sclerosis (MS). Here, we evaluated the astrocyte contribution to myelin loss and focused on the neurotrophin receptor TrkB, whose up-regulation on the astrocyte finely demarcated chronic demyelinated areas in MS and was paralleled by neurotrophin loss. Mice lacking astrocyte TrkB were resistant to demyelination induced by autoimmune or toxic insults, demonstrating that TrkB signaling in astrocytes fostered oligodendrocyte damage. In vitro and ex vivo approaches highlighted that astrocyte TrkB supported scar formation and glia proliferation even in the absence of neurotrophin binding, indicating TrkB transactivation in response to inflammatory or toxic mediators. Notably, our neuropathological studies demonstrated copper dysregulation in MS and model lesions and TrkB-dependent expression of copper transporter (CTR1) on glia cells during neuroinflammation. In vitro experiments evidenced that TrkB was critical for the generation of glial intracellular calcium flux and CTR1 up-regulation induced by stimuli distinct from neurotrophins. These events led to copper uptake and release by the astrocyte, and in turn resulted in oligodendrocyte loss. Collectively, these data demonstrate a pathogenic demyelination mechanism via the astrocyte release of copper and open up the possibility of restoring copper homeostasis in the white matter as a therapeutic target in MS.

Reactive astrocyte nomenclature, definitions, and future directions.

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions.

The phenotypic convergence between microglia and peripheral macrophages during development and neuroinflammation paves the way for new therapeutic perspectives.

Microglia, the tissue resident macrophages of the brain, are increasingly recognized as key players for central nervous system development and homeostasis. They are long-lived cells deriving from a transient wave of yolk-sac derived erythro-myeloid progenitors early in development. Their unique ontology has prompted the search for specific markers to be used for their selective investigation and manipulation. The first generation of genome-wide expression studies has provided a bundle of transcripts (such as Olfml3, Fcrls, Tmem119, P2ry12, Gpr34, and Siglech) useful to distinguish microglia from peripheral macrophages. However, more recent reports have revealed that microglial phenotype is constantly shaped by the microenvironment in a time-, and context-dependent manner. In this article, we review data that provide additional pieces to this complex scenario and show the existence of unexpected phenotypic convergence between microglia and peripheral macrophages at certain developmental stages and under pathological conditions. These observations suggest that the two cell types act synergically boosting their mutual activities depending on the microenvironment. This novel information about the biology of microglia and peripheral macrophages sheds new light about their therapeutic potential for neuroinflammatory and neurodegenerative diseases.

Laquinimod Modulates Human Astrocyte Function and Dampens Astrocyte-Induced Neurotoxicity during Inflammation.

Astrocytes greatly participate to inflammatory and neurotoxic reactions occurring in neurodegenerative diseases and are valuable pharmacological targets to support neuroprotection. Here we used human astrocytes generated from reprogrammed fibroblasts as a cellular model to study the effect of the compound Laquinimod and its active metabolite de-Laquinimod on astrocyte functions and the astrocyte-neuron interaction. We show that human iAstrocytes expressed the receptor for the inflammatory mediator IL1 and responded to it via nuclear translocation of NFκB, an event that did not occur if cells were treated with Laquinimod, indicating a direct anti-inflammatory activity of the drug on the human astrocyte. Similarly, while exposure to IL1 downregulated glial glutamate transporters GLAST and GLT1, treatment with Laquinimod supported maintenance of physiological levels of these proteins despite the inflammatory milieu. Laquinimod also induced nuclear translocation of the aryl hydrocarbon receptor (AHR), suggesting that drug action was mediated by activation of the AHR pathway. However, the drug was effective despite AHR inhibition via CH223191, indicating that AHR signaling in the astrocyte is dispensable for drug responses. Finally, in vitro experiments with rat spinal neurons showed that laquinimod did not exert neuroprotection directly on the neuron but dampened astrocyte-induced neurodegeneration. Our findings indicate that fibroblast-derived human astrocytes represent a suitable model to study astrocyte-neuron crosstalk and demonstrate indirect, partial neuroprotective efficacy for laquinimod.

Inferring Multiple Sclerosis Stages from the Blood Transcriptome via Machine Learning.

Peripheral blood mononuclear cells (PBMCs) bear specific dysregulations in genes and pathways at distinct stages of multiple sclerosis (MS) that may help with classifying MS and non-MS subjects, specifying the early stage of disease, or discriminating among MS courses. Here we describe an unbiased machine learning workflow to build MS stage-specific classifiers based on PBMC transcriptomics profiles from more than 300 individuals, including healthy subjects and patients with clinically isolated syndromes, relapsing-remitting MS, primary or secondary progressive MS, or other neurological disorders. The pipeline, designed to optimize and compare the performance of distinct machine learning algorithms in the training cohort, generates predictive models not influenced by demographic features, such as age and gender, and displays high accuracy in the independent validation cohort. Proper application of machine learning to transcriptional profiles of circulating blood cells may allow identification of disease state and stage in MS.

Immune profiling of plasma-derived extracellular vesicles identifies Parkinson disease.

OBJECTIVE: To develop a diagnostic model based on plasma-derived extracellular vesicle (EV) subpopulations in Parkinson disease (PD) and atypical parkinsonism (AP), we applied an innovative flow cytometric multiplex bead-based platform. METHODS: Plasma-derived EVs were isolated from PD, matched healthy controls, multiple system atrophy (MSA), and AP with tauopathies (AP-Tau). The expression levels of 37 EV surface markers were measured by flow cytometry and correlated with clinical scales. A diagnostic model based on EV surface markers expression was built via supervised machine learning algorithms and validated in an external cohort. RESULTS: Distinctive pools of EV surface markers related to inflammatory and immune cells stratified patients according to the clinical diagnosis. PD and MSA displayed a greater pool of overexpressed immune markers, suggesting a different immune dysregulation in PD and MSA vs AP-Tau. The receiver operating characteristic curve analysis of a compound EV marker showed optimal diagnostic performance for PD (area under the curve [AUC] 0.908; sensitivity 96.3%, specificity 78.9%) and MSA (AUC 0.974; sensitivity 100%, specificity 94.7%) and good accuracy for AP-Tau (AUC 0.718; sensitivity 77.8%, specificity 89.5%). A diagnostic model based on EV marker expression correctly classified 88.9% of patients with reliable diagnostic performance after internal and external validations. CONCLUSIONS: Immune profiling of plasmatic EVs represents a crucial step toward the identification of biomarkers of disease for PD and AP.

Siponimod (BAF312) Activates Nrf2 While Hampering NFκB in Human Astrocytes, and Protects From Astrocyte-Induced Neurodegeneration.

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) with heterogeneous pathophysiology. In its progressive course oligodendrocyte and neuroaxonal damage is sustained by compartmentalized inflammation due to glial dysregulation. Siponimod (BAF312), a modulator of two sphingosine-1-phosphate (S1P) receptors (S1P1 and S1P5) is the first oral treatment specifically approved for active secondary progressive MS. To address potential direct effects of BAF312 on glial function and glia-neuron interaction, we set up a series of in vitro functional assays with astrocytes generated from human fibroblasts. These cells displayed the typical morphology and markers of astroglia, and were susceptible to the action of inflammatory mediators and BAF312, because expressing receptors for IL1, IL17, and S1P (namely S1P1 and S1P3). Targeting of S1P signaling by BAF312 inhibited NFκB translocation evoked by inflammatory cytokines, indicating a direct anti-inflammatory activity of the drug on the human astrocyte. Further, while glia cells exposed to IL1 or IL17 downregulated protein expression of glutamate transporters, BAF312-treated astrocytes maintained high levels of GLAST and GLT1 regardless of the presence of inflammatory mediators. Interestingly, despite potential glial susceptibility to S1P signaling via S1P3, which is not targeted by BAF312, NFκB translocation and downregulation of glutamate transporters in response to S1P were inhibited at similar levels by BAF312 and FTY720, another S1P signaling modulator targeting also S1P3. Accordingly, specific inhibition of S1P1 via NIBR-0213 blocked S1P-evoked NFκB translocation, demonstrating that modulation of S1P1 is sufficient to dampen signaling via other S1P receptors. Considering that NFκB-dependent responses are regulated by Nrf2, we measured activation of this critical transcription factor for anti-oxidant reactions, and observed that BAF312 rapidly induced nuclear translocation of Nrf2, but this effect was attenuated in the presence of an inflammatory milieu. Finally, in vitro experiments with spinal neurons exposed to astrocyte-conditioned media showed that modulation of S1P or cytokine signaling in astrocytes via BAF312 prevented neurons from astrocyte-induced degeneration. Overall, these experiments on human astrocytes suggest that during neuroinflammation targeting of S1P1 via BAF312 may modulate key astrocyte functions and thereby attain neuroprotection indirectly.

Convergence between Microglia and Peripheral Macrophages Phenotype during Development and Neuroinflammation.

Differently from other myeloid cells, microglia derive exclusively from precursors originating within the yolk sac and migrate to the CNS under development, without any contribution from fetal liver or postnatal hematopoiesis. Consistent with their unique ontology, microglia may express specific physiological markers, which have been partly described in recent years. Here we wondered whether profiles distinguishing microglia from peripheral macrophages vary with age and under pathology. To this goal, we profiled transcriptomes of microglia throughout the lifespan and included a parallel comparison with peripheral macrophages under physiological and neuroinflammatory settings using age- and sex-matched wild-type and bone marrow chimera mouse models. This comprehensive approach demonstrated that the phenotypic differentiation between microglia and peripheral macrophages is age-dependent and that peripheral macrophages do express some of the most commonly described microglia-specific markers early during development, such as Fcrls, P2ry12, Tmem119, and Trem2. Further, during chronic neuroinflammation CNS-infiltrating macrophages and not peripheral myeloid cells acquire microglial markers, indicating that the CNS niche may instruct peripheral myeloid cells to gain the phenotype and, presumably, the function of the microglia cell. In conclusion, our data provide further evidence about the plasticity of the myeloid cell and suggest caution in the strict definition and application of microglia-specific markers.SIGNIFICANCE STATEMENT Understanding the respective role of microglia and infiltrating monocytes in neuroinflammatory conditions has recently seemed possible by the identification of a specific microglia signature. Here instead we provide evidence that peripheral macrophages may express some of the most commonly described microglia markers at some developmental stages or pathological conditions, in particular during chronic neuroinflammation. Further, our data support the hypothesis about phenotypic plasticity and convergence among distinct myeloid cells so that they may act as a functional unit rather than as different entities, boosting their mutual functions in different phases of disease. This holds relevant implications in the view of the growing use of myeloid cell therapies to treat brain disease in humans.

Loss of Circulating CD8+ CD161high T Cells in Primary Progressive Multiple Sclerosis.

Recent evidence suggests that the primary progressive form of multiple sclerosis (PP-MS) may present with specific immunological alterations. In this study we focused our attention on CD161, an NK and T cell marker upregulated in relapsing-remitting MS, and investigated its transcript and protein levels in blood cells from PP-MS and healthy individuals. We demonstrated transcriptional downregulation of CD161 in PP-MS and described concomitant mRNA reduction for RORgt, CCR6, CXCR6, KLRK1/NKG2D and many other markers typical of mucosa associated invariant T (MAIT) cells. Targeted multiparametric flow cytometry on fresh blood cells from an independent cohort of case-control subjects confirmed the selective loss of circulating CD8 CD161high T cells, which consist mainly of MAIT cells, and not of CD8 CD161int T cells in PP-MS. These data demonstrate alterations in a specific circulating immune cell subset in MS patients with progressive onset.

A cell type-specific transcriptomic approach to map B cell and monocyte type I interferon-linked pathogenic signatures in Multiple Sclerosis.

Alteration in endogenous Interferon (IFN) system may profoundly impact immune cell function in autoimmune diseases. Here, we provide evidence that dysregulation in IFN-regulated genes and pathways are involved in B cell- and monocyte-driven pathogenic contribution to Multiple Sclerosis (MS) development and maintenance. In particular, by using an Interferome-based cell type-specific approach, we characterized an increased susceptibility to an IFN-linked caspase-3 dependent apoptotic cell death in both B cells and monocytes of MS patients that may arise from their chronic activation and persistent stimulation by activated T cells. Ongoing caspase-3 activation functionally impacts on MS monocyte properties influencing the STAT-3/IL-16 axis, thus, driving increased expression and massive release of the bio-active IL-16 triggering and perpetuating CD4+ T cell migration. Importantly, our analysis also identified a previously unknown multi-component defect in type I IFN-mediated signaling and response to virus pathways specific of MS B cells, impacting on induction of anti-viral responses and Epstein-barr virus infection control in patients. Taking advantage of cell type-specific transcriptomics and in-depth functional validation, this study revealed pathogenic contribution of endogenous IFN signaling and IFN-regulated cell processes to MS pathogenesis with implications on fate and functions of B cells and monocytes that may hold therapeutic potential.

Author Correction: Transcriptional dysregulation of Interferome in experimental and human Multiple Sclerosis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.