Physiological aging and inflammation-induced cellular senescence may contribute to oligodendroglial dysfunction in MS.

Aging affects all cell types in the CNS and plays an important role in CNS diseases. However, the underlying molecular mechanisms driving these age-associated changes and their contribution to diseases are only poorly understood. The white matter in the aging brain as well as in diseases, such as Multiple sclerosis is characterized by subtle abnormalities in myelin sheaths and paranodes, suggesting that oligodendrocytes, the myelin-maintaining cells of the CNS, lose the capacity to preserve a proper myelin structure and potentially function in age and certain diseases. Here, we made use of directly converted oligodendrocytes (dchiOL) from young, adult and old human donors to study age-associated changes. dchiOL from all three age groups differentiated in an comparable manner into O4 + immature oligodendrocytes, but the proportion of MBP + mature dchiOL decreased with increasing donor age. This was associated with an increased ROS production and upregulation of cellular senescence markers such as CDKN1A, CDKN2A in old dchiOL. Comparison of the transcriptomic profiles of dchiOL from adult and old donors revealed 1324 differentially regulated genes with limited overlap with transcriptomic profiles of the donors' fibroblasts or published data sets from directly converted human neurons or primary rodent oligodendroglial lineage cells. Methylome analyses of dchiOL and human white matter tissue samples demonstrate that chronological and epigenetic age correlate in CNS white matter as well as in dchiOL and resulted in the identification of an age-specific epigenetic signature. Furthermore, we observed an accelerated epigenetic aging of the myelinated, normal appearing white matter of multiple sclerosis (MS) patients compared to healthy individuals. Impaired differentiation and upregulation of cellular senescence markers could be induced in young dchiOL in vitro using supernatants from pro-inflammatory microglia. In summary, our data suggest that physiological aging as well as inflammation-induced cellular senescence contribute to oligodendroglial pathology in inflammatory demyelinating diseases such as MS.

An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders.

BACKGROUND: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. METHODS: Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. RESULTS: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls. CONCLUSIONS: We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.

Age-dependent effects of metformin on human oligodendrocyte lineage cell ensheathment capacity.

Metformin restores the myelination potential of aged rat A2B5+ oligodendrocyte progenitor cells and may enhance recovery in children with post-radiation brain injury. Human late progenitor cells (O4+A2B5+) have a superior capacity to ensheath nanofibres compared to mature oligodendrocytes, with cells from paediatric sources exceeding adults. In this study, we assessed the effects of metformin on ensheathment capacity of human adult and paediatric progenitors and mature oligodendrocytes and related differences to transcriptional changes. A2B5+ progenitors and mature cells, derived from surgical tissues by immune-magnetic separation, were assessed for ensheathment capacity in nanofibre plates over 2 weeks. Metformin (10 µM every other day) was added to selected cultures. RNA was extracted from treated and control cultures after 2 days. For all ages, ensheathment by progenitors exceeded mature oligodendrocytes. Metformin enhanced ensheathment by adult donor cells but reduced ensheathment by paediatric cells. Metformin marginally increased cell death in paediatric progenitors. Metformin-induced changes in gene expression are distinct for each cell type. Adult progenitors showed up-regulation of pathways involved in the process of outgrowth and promoting lipid biosynthesis. Paediatric progenitors showed a relatively greater proportion of down- versus up-regulated pathways, these involved cell morphology, development and synaptic transmission. Metformin-induced AMP-activated protein kinase activation in all cell types; AMP-activated protein kinase inhibitor BML-275 reduced functional metformin effects only with adult cells. Our results indicate age and differentiation stage-related differences in human oligodendroglia lineage cells in response to metformin. Clinical trials for demyelinating conditions will indicate how these differences translate in vivo.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Identity and nature of neural stem cells in the adult human subventricular zone.

The existence of neural stem cells (NSCs) in adult human brain neurogenic regions remains unresolved. To address this, we created a cell atlas of the adult human subventricular zone (SVZ) derived from fresh neurosurgical samples using single-cell transcriptomics. We discovered 2 adult radial glia (RG)-like populations, aRG1 and aRG2. aRG1 shared features with fetal early RG (eRG) and aRG2 were transcriptomically similar to fetal outer RG (oRG). We also captured early neuronal and oligodendrocytic NSC states. We found that the biological programs driven by their transcriptomes support their roles as early lineage NSCs. Finally, we show that these NSCs have the potential to transition between states and along lineage trajectories. These data reveal that multipotent NSCs reside in the adult human SVZ.

The use of a SOX10 reporter toward ameliorating oligodendrocyte lineage differentiation from human induced pluripotent stem cells.

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor-based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10-P2A-mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells. Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, toward enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but also holds promise to accelerate research and translational work with iPSC-derived OLs.

Regulation of stress granule formation in human oligodendrocytes.

Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Astrocytes play important roles in the central nervous system (CNS) physiology and pathology. Indeed, astrocyte subsets defined by specific transcriptional activation states contribute to the pathology of neurologic diseases, including multiple sclerosis (MS) and its pre-clinical model experimental autoimmune encephalomyelitis (EAE) 1-8 . However, little is known about the stability of these disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges. Here, we describe the identification of an epigenetically controlled memory astrocyte subset which exhibits exacerbated pro-inflammatory responses upon re-challenge. Specifically, using a combination of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), and cell-specific in vivo CRISPR/Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) used by the histone acetyltransferase p300 to control chromatin accessibility. ACLY + p300 + memory astrocytes are increased in acute and chronic EAE models; the genetic targeting of ACLY + p300 + astrocytes using CRISPR/Cas9 ameliorated EAE. We also detected responses consistent with a pro-inflammatory memory phenotype in human astrocytes in vitro ; scRNA-seq and immunohistochemistry studies detected increased ACLY + p300 + astrocytes in chronic MS lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, MS. These findings may guide novel therapeutic approaches for MS and other neurologic diseases.

ALCAM on human oligodendrocytes mediates CD4 T cell adhesion.

Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the CNS. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 Th17 cells can interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we used single cell RNA sequencing, flow cytometry and immunofluorescence studies on CNS tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM) in multiple sclerosis, in experimental autoimmune encephalomyelitis and in controls, although their regulation differs between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of MCAM but not of ALCAM at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live imaging, immunofluorescence and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking ALCAM but not MCAM limited prolonged interactions between human primary oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferring significant protection of oligodendrocytic processes. In conclusion, we showed that human oligodendrocytes express MCAM and ALCAM, which are differently modulated by inflammation and T cell contact. We found that ALCAM is a ligand for Th17-polarized cells, contributing to their capacity to adhere and induce damage to human oligodendrocytes, and therefore could represent a relevant target for neuroprotection in multiple sclerosis.

MerTK is a mediator of alpha-synuclein fibril uptake by human microglia.

Mer tyrosine kinase (MerTK) is a receptor tyrosine kinase that mediates non-inflammatory, homeostatic phagocytosis of diverse types of cellular debris. Highly expressed on the surface of microglial cells, MerTK is of importance in brain development, homeostasis, plasticity and disease. Yet, involvement of this receptor in the clearance of protein aggregates that accumulate with ageing and in neurodegenerative diseases has yet to be defined. The current study explored the function of MerTK in the microglial uptake of alpha-synuclein fibrils which play a causative role in the pathobiology of synucleinopathies. Using human primary and induced pluripotent stem cell-derived microglia, the MerTK-dependence of alpha-synuclein fibril internalization was investigated in vitro. Relevance of this pathway in synucleinopathies was assessed through burden analysis of MERTK variants and analysis of MerTK expression in patient-derived cells and tissues. Pharmacological inhibition of MerTK and siRNA-mediated MERTK knockdown both caused a decreased rate of alpha-synuclein fibril internalization by human microglia. Consistent with the non-inflammatory nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization was not observed to induce secretion of pro-inflammatory cytokines such as IL-6 or TNF, and downmodulated IL-1ß secretion from microglia. Burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinson's disease in one of the cohorts (P = 0.002). Despite a small upregulation in MERTK mRNA expression in nigral microglia from Parkinson's disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (P = 5.08 × 10-21), no significant upregulation in MerTK protein expression was observed in human cortex and substantia nigra lysates from Lewy body dementia patients compared to controls. Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein fibrils by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinson's disease. Upregulation of this pathway in synucleinopathies could have therapeutic values in enhancing alpha-synuclein fibril clearance in the brain.

Cross-talk between B cells, microglia and macrophages, and implications to central nervous system compartmentalized inflammation and progressive multiple sclerosis.

BACKGROUND: B cells can be enriched within meningeal immune-cell aggregates of multiple sclerosis (MS) patients, adjacent to subpial cortical demyelinating lesions now recognized as important contributors to progressive disease. This subpial demyelination is notable for a 'surface-in' gradient of neuronal loss and microglial activation, potentially reflecting the effects of soluble factors secreted into the CSF. We previously demonstrated that MS B-cell secreted products are toxic to oligodendrocytes and neurons. The potential for B-cell-myeloid cell interactions to propagate progressive MS is of considerable interest. METHODS: Secreted products of MS-implicated pro-inflammatory effector B cells or IL-10-expressing B cells with regulatory potential were applied to human brain-derived microglia or monocyte-derived macrophages, with subsequent assessment of myeloid phenotype and function through measurement of their expression of pro-inflammatory, anti-inflammatory and homeostatic/quiescent molecules, and phagocytosis (using flow cytometry, ELISA and fluorescently-labeled myelin). Effects of secreted products of differentially activated microglia on B-cell survival and activation were further studied. FINDINGS: Secreted products of MS-implicated pro-inflammatory B cells (but not IL-10 expressing B cells) substantially induce pro-inflammatory cytokine (IL-12, IL-6, TNFα) expression by both human microglia and macrophage (in a GM-CSF dependent manner), while down-regulating their expression of IL-10 and of quiescence-associated molecules, and suppressing their myelin phagocytosis. In contrast, secreted products of IL-10 expressing B cells upregulate both human microglia and macrophage expression of quiescence-associated molecules and enhance their myelin phagocytosis. Secreted factors from pro-inflammatory microglia enhance B-cell activation. INTERPRETATION: Potential cross-talk between disease-relevant human B-cell subsets and both resident CNS microglia and infiltrating macrophages may propagate CNS-compartmentalized inflammation and injury associated with MS disease progression. These interaction represents an attractive therapeutic target for agents such as Bruton's tyrosine kinase inhibitors (BTKi) that modulate responses of both B cells and myeloid cells. FUNDING: Stated in Acknowledgments section of manuscript.

Translocator protein is a marker of activated microglia in rodent models but not human neurodegenerative diseases.

Microglial activation plays central roles in neuroinflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18 kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells depends on the transcription factor AP1 and is unique to a subset of rodent species within the Muroidea superfamily. Finally, we identify LCP2 and TFEC as potential markers of microglial activation in humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO-PET signals in humans reflect the density of inflammatory cells rather than activation state.

Mechanisms of metabolic stress induced cell death of human oligodendrocytes: relevance for progressive multiple sclerosis.

Oligodendrocyte (OL) injury and loss are central features of evolving lesions in multiple sclerosis. Potential causative mechanisms of OL loss include metabolic stress within the lesion microenvironment. Here we use the injury response of primary human OLs (hOLs) to metabolic stress (reduced glucose/nutrients) in vitro to help define the basis for the in situ features of OLs in cases of MS. Under metabolic stress in vitro, we detected reduction in ATP levels per cell that precede changes in survival. Autophagy was initially activated, although ATP levels were not altered by inhibitors (chloroquine) or activators (Torin-1). Prolonged stress resulted in autophagy failure, documented by non-fusion of autophagosomes and lysosomes. Consistent with our in vitro results, we detected higher expression of LC3, a marker of autophagosomes in OLs, in MS lesions compared to controls. Both in vitro and in situ, we observe a reduction in nuclear size of remaining OLs. Prolonged stress resulted in increased ROS and cleavage of spectrin, a target of Ca2+-dependent proteases. Cell death was however not prevented by inhibitors of ferroptosis or MPT-driven necrosis, the regulated cell death (RCD) pathways most likely to be activated by metabolic stress. hOLs have decreased expression of VDAC1, VDAC2, and of genes regulating iron accumulation and cyclophilin. RNA sequencing analyses did not identify activation of these RCD pathways in vitro or in MS cases. We conclude that this distinct response of hOLs, including resistance to RCD, reflects the combined impact of autophagy failure, increased ROS, and calcium influx, resulting in metabolic collapse and degeneration of cellular structural integrity. Defining the basis of OL injury and death provides guidance for development of neuro-protective strategies.

Multiple sclerosis: 2023 update.

Multiple sclerosis (MS) is the most frequent inflammatory and demyelinating disease of the Central Nervous System (CNS). Significant progress has been made during recent years in preventing relapses by using systemic immunomodulatory or immunosuppressive therapies. However, the limited effectiveness of such therapies for controlling the progressive disease course indicates there is a continuous disease progression independent of relapse activity which may start very early during the disease course. Dissecting the underlying mechanisms and developing therapies for preventing or stopping this disease progression represent, currently, the biggest challenges in the field of MS. Here, we summarize publications of 2022 which provide insight into susceptibility to MS, the basis of disease progression and features of relatively recently recognized distinct forms of inflammatory/demyelinating disorders of the CNS, such as myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD).

Clinical course, imaging, and pathological features of 45 adult and pediatric cases of myelin oligodendrocyte glycoprotein antibody-associated disease.

BACKGROUND: Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is a recently described neuroinflammatory demyelinating disease. OBJECTIVE: To better understand the clinical spectrum, risk factors and outcomes in MOGAD. METHODS: Retrospective cohort study including all subjects harboring anti-MOG antibodies identified in major academic hospitals across the province of Quebec. RESULTS: We identified 45 MOGAD cases. The minimal estimated point-prevalence was 0.52/100 000 in Quebec. Median age at presentation was 32 years (range 1-71) with equal sex ratio. Most frequent ethnic groups were Caucasians and Asians. The most frequent clinical manifestations at onset were optic neuritis (ON), affecting 56% of adults, and acute disseminated encephalomyelitis (ADEM), affecting 33% of children. First MRI was abnormal in 84% of cases. Most CSF samples showed pleocytosis without oligoclonal bands. Two brain biopsies revealed lipid-laden macrophages and reactive astrocytes. Despite steroids, only 38% had fully recovered at 4 weeks after onset. Half of pediatric and two thirds of adult-onset MOGAD subjects experienced relapses. At last follow-up, 69% showed residual deficits, which were moderate to severe in 17% of adults. CONCLUSION: MOGAD has heterogeneous disease course, and it is not a benign disease for a substantial proportion of adults. Best disease-modifying therapies remain to be determined.

Analysis of the microglia transcriptome across the human lifespan using single cell RNA sequencing.

BACKGROUND: Microglia are tissue resident macrophages with a wide range of critically important functions in central nervous system development and homeostasis. METHOD: In this study, we aimed to characterize the transcriptional landscape of ex vivo human microglia across different developmental ages using cells derived from pre-natal, pediatric, adolescent, and adult brain samples. We further confirmed our transcriptional observations using ELISA and RNAscope. RESULTS: We showed that pre-natal microglia have a distinct transcriptional and regulatory signature relative to their post-natal counterparts that includes an upregulation of phagocytic pathways. We confirmed upregulation of CD36, a positive regulator of phagocytosis, in pre-natal samples compared to adult samples in situ. Moreover, we showed adult microglia have more pro-inflammatory signature compared to microglia from other developmental ages. We indicated that adult microglia are more immune responsive by secreting increased levels of pro-inflammatory cytokines in response to LPS treatment compared to the pre-natal microglia. We further validated in situ up-regulation of IL18 and CXCR4 in human adult brain section compared to the pre-natal brain section. Finally, trajectory analysis indicated that the transcriptional signatures adopted by microglia throughout development are in response to a changing brain microenvironment and do not reflect predetermined developmental states. CONCLUSION: In all, this study provides unique insight into the development of human microglia and a useful reference for understanding microglial contribution to developmental and age-related human disease.

Seeking neuroprotection in multiple sclerosis: an ongoing challenge.

Multiple sclerosis (MS) is an autoimmune disease of the CNS, featuring inflammation and demyelination with variable recovery. In this issue of the JCI, Kapell, Fazio, and authors address the potential for targeting neuron-oligodendrocyte potassium shuttling at the nodes of Ranvier as a neuroprotective strategy during inflammatory demyelination of the CNS in experimental MS. Their extensive and impressive study could serve as a template for defining the physiologic properties of a putative protective pathway. The authors examined MS features in existent disease models, investigated the impact of pharmacologic intervention, and evaluated its status in tissues from patients with MS. We await future studies that will tackle the challenge of translating these findings into a clinical therapy.

Inflammation in multiple sclerosis: consequences for remyelination and disease progression.

Despite the large number of immunomodulatory or immunosuppressive treatments available to treat relapsing-remitting multiple sclerosis (MS), treatment of the progressive phase of the disease has not yet been achieved. This lack of successful treatment approaches is caused by our poor understanding of the mechanisms driving disease progression. Emerging concepts suggest that a combination of persisting focal and diffuse inflammation within the CNS and a gradual failure of compensatory mechanisms, including remyelination, result in disease progression. Therefore, promotion of remyelination presents a promising intervention approach. However, despite our increasing knowledge regarding the cellular and molecular mechanisms regulating remyelination in animal models, therapeutic increases in remyelination remain an unmet need in MS, which suggests that mechanisms of remyelination and remyelination failure differ fundamentally between humans and demyelinating animal models. New and emerging technologies now allow us to investigate the cellular and molecular mechanisms underlying remyelination failure in human tissue samples in an unprecedented way. The aim of this Review is to summarize our current knowledge regarding mechanisms of remyelination and remyelination failure in MS and in animal models of the disease, identify open questions, challenge existing concepts, and discuss strategies to overcome the translational roadblock in the field of remyelination-promoting therapies.

Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Systematic comparison of culture media uncovers phenotypic shift of primary human microglia defined by reduced reliance to CSF1R signaling.

Efforts to understand microglia function in health and diseases have been hindered by the lack of culture models that recapitulate in situ cellular properties. In recent years, the use of serum-free media with brain-derived growth factors (colony stimulating factor 1 receptor [CSF1R] ligands and TGF-ß1/2) have been favored for the maintenance of rodent microglia as they promote morphological features observed in situ. Here we study the functional and transcriptomic impacts of such media on human microglia (hMGL). Media formulation had little impact on microglia transcriptome assessed by RNA sequencing which was sufficient to significantly alter microglia capacity to phagocytose myelin debris and to elicit an inflammatory response to lipopolysaccharide. When compared to immediately ex vivo microglia from the same donors, the addition of fetal bovine serum to culture media, but not growth factors, was found to aid in the maintenance of key signature genes including those involved in phagocytic processes. A phenotypic shift characterized by CSF1R downregulation in culture correlated with a lack of reliance on CSF1R signaling for survival. Consequently, no improvement in cell survival was observed following culture supplementation with CSF1R ligands. Our study provides better understanding of hMGL in culture, with observations that diverge from those previously made in rodent microglia.