Neural stem cells (NSCs) in the subventricular zone (SVZ) of the mammalian brain become increasingly quiescent with aging, which correlates to increased inflammatory signals in the SVZ. Targeting cells that secrete inflammatory signals, such as microglia, could potentially re-activate NSCs. In this study, we characterized CD11b-positive microglia isolated from post-mortem SVZ from non-demented control (Aged), Alzheimer's disease (AD), and Parkinson's disease (PD) by single-cell and bulk RNA sequencing. Our transcriptome data revealed changes in gene signature in SVZ microglia from PD and AD, highlighting a disease-dependent response. Culture of iPSC-derived NSCs with supernatant from Aged, PD, and AD SVZ microglia showed an increase in proliferation and neuronal differentiation in the PD condition. Furthermore, we identified NR4A2, a transcription factor that promotes an anti-inflammatory microglia state, as a potential molecular mechanism that promotes a pro-neurogenic microglia phenotype. Altogether, our work identified a pro-neurogenic subpopulation of SVZ microglia that could be a novel target to promote repair in neurodegenerative diseases.

Neurogenesis continues throughout adulthood in specialized regions of the brain. One of these regions is the subventricular zone. During brain development, neurogenesis is regulated by a complex interplay of intrinsic and extrinsic cues that control stem-cell survival, renewal and cell lineage specification. Cerebrospinal fluid (CSF) is an integral part of the neurogenic niche in development as it is in direct contact with radial glial cells, and it is important in regulating proliferation and migration. Yet, the effect of CSF on neural stem cells in the subventricular zone of the adult human brain is unknown. We hypothesized a persistent stimulating effect of ventricular CSF on neural stem cells in adulthood, based on the literature, describing bulging accumulations of subventricular cells where CSF is in direct contact with the subventricular zone. Here, we show by immunohistochemistry on post-mortem adult human subventricular zone sections that neural stem cells are in close contact with CSF via protrusions through both intact and incomplete ependymal layers. We are the first to systematically quantify subventricular glial nodules denuded of ependyma and consisting of proliferating neural stem and progenitor cells, and showed that they are present from foetal age until adulthood. Neurosphere, cell motility and differentiation assays as well as analyses of RNA expression were used to assess the effects of CSF of adult humans on primary neural stem cells and a human immortalized neural stem cell line. We show that human ventricular CSF increases proliferation and decreases motility of neural stem cells. Our results also indicate that adult CSF pushes neural stem cells from a relative quiescent to a more active state and promotes neuronal over astrocytic lineage differentiation. Thus, CSF continues to stimulate neural stem cells throughout aging.

It is currently accepted that the human brain has a limited neurogenic capacity and an impaired regenerative potential. We have previously shown the existence of CD271-expressing neural stem cells (NSCs) in the subventricular zone (SVZ) of Parkinson’s disease (PD) patients, which proliferate and differentiate towards neurons and glial cells in vitro. To study the molecular profile of these NSCs in detail, we performed RNA sequencing and mass spectrometry on CD271 + NSCs isolated from human post-mortem SVZ and on homogenates of the SVZ. CD271 + cells were isolated through magnetic cell separation (MACS). We first compared the molecular profile of CD271 + NSCs to the SVZ homogenate from control donors and then compared CD271 + cells to CD11b + microglia. These results confirmed their neural stem cell identity. Finally we compared controls and PD patients to establish a specific molecular profile of NSCs and the SVZ in PD. While our transcriptome analysis did not identify any differentially expressed genes in the SVZ between control and PD patients, our proteome analysis revealed several proteins that were differentially expressed in PD. Some of these proteins are involved in cytoskeletal organization and mitochondrial function. Transcriptome and proteome analyses of NSCs from PD revealed changes in the expression of genes and proteins involved in metabolism, transcriptional activity and cytoskeletal organization. Our data suggest that NSCs may transit into a primed-quiescent state, that is in an “alert” non-proliferative phase in PD. Our results not only confirm pathological hallmarks of PD (e.g. impaired mitochondrial function), but also show that the NSCs from SVZ undergo significant changes at both transcriptome and proteome level following PD.

Gliomas are the most common primary brain tumors. Their highly invasive character and the heterogeneity of active oncogenic pathways within single tumors complicate the development of curative therapies and cause poor patient prognosis. Glioma cells express the intermediate filament protein glial fibrillary acidic protein (GFAP), and the level of its alternative splice variant GFAP‐δ, relative to its canonical splice variant GFAP‐α, is higher in grade IV compared with lower‐grade and lower malignant glioma. In this study we show that a high GFAP‐δ/α ratio induces the expression of the dual‐specificity phosphatase 4 (DUSP4) in focal adhesions. By focusing on pathways up‐ and downstream of DUSP4 that are involved in the cell‐extracellular matrix interaction, we show that a high GFAP‐δ/α ratio equips glioma cells to better invade the brain. This study supports the hypothesis that glioma cells with a high GFAP‐δ/α ratio are highly invasive and more malignant cells, thus making GFAP alternative splicing a potential therapeutic target.—Van Bodegraven, E. J., van Asperen, J. V., Sluijs, J. A., van Deursen, C. B. J., van Strien, M. E., Stassen, O. M. J. A., Robe, P. A. J., Hol, E. M. GFAP alternative splicing regulates glioma cell–ECM interaction in a DUSP4‐dependent manner. FASEB J. 33, 12941–12959 (2019). www.fasebj.org

Astrocytomas are the most common malignant brain tumours and are to date incurable. It is unclear how astrocytomas progress into higher malignant grades. The intermediate filament cytoskeleton is emerging as an important regulator of malignancy in several tumours. The majority of the astrocytomas express the intermediate filament protein Glial Fibrillary Acidic Protein (GFAP). Several GFAP splice variants have been identified and the main variants expressed in human astrocytoma are the GFAPα and GFAPδ isoforms. Here we show a significant downregulation of GFAPα in grade IV astrocytoma compared to grade II and III, resulting in an increased GFAPδ/α ratio. Mimicking this increase in GFAPδ/α ratio in astrocytoma cell lines and comparing the subsequent transcriptomic changes with the changes in the patient tumours, we have identified a set of GFAPδ/α ratio-regulated high-malignant and low-malignant genes. These genes are involved in cell proliferation and protein phosphorylation, and their expression correlated with patient survival. We additionally show that changing the ratio of GFAPδ/α, by targeting GFAP expression, affected expression of high-malignant genes. Our data imply that regulating GFAP expression and splicing are novel therapeutic targets that need to be considered as a treatment for astrocytoma.

Genetic, epidemiological and post mortem studies have described an association between schizophrenia (SCZ) and the immune system. Microglia, the tissue-resident macrophages of the brain, not only play an essential role in inflammatory processes, but also in neurodevelopment and synapse refinement. It has therefore been hypothesized that aberrant functioning of these myeloid immune cells is involved in SCZ pathogenesis. Until now cellular research into the role of myeloid cells in SCZ has been limited to monocytes and functional assays are lacking. In this study we used monocyte-derived macrophages (mo-MΦs) as a model for macrophages and microglia in the CNS and examined two main functions: Inflammatory responses and expression and regulation of synapse refinement molecules. The expression of 24 genes involved in these key functions was assessed. Mo-MΦs were generated from 15 SCZ patients and 15 healthy controls. The cells were exposed to pro-inflammatory and anti-inflammatory stimuli (LPS, R848, IL-4 and dexamethasone), and the response was measured by qPCR and ELISA analyses. One of the genes of interest, P2RX7 that is associated with psychiatric diseases, was significantly reduced in expression after LPS stimulation in SCZ patients. None of the other assessed characteristics were different in this functional screen between mo-MΦs from SCZ patients compared to controls. Although these data suggest that overall the function of macrophages in SCZ is not impaired, further studies with larger groups that enable the possibility to study clinical subgroups and perform additional screenings to asses the full phenotype of the mo-MΦs are needed to strengthen this conclusion.

Microglia are key players in the central nervous system in health and disease. Much pioneering research on microglia function has been carried out in vivo with the use of genetic animal models. However, to fully understand the role of microglia in neurological and psychiatric disorders, it is crucial to study primary human microglia from brain donors. We have developed a rapid procedure for the isolation of pure human microglia from autopsy tissue using density gradient centrifugation followed by CD11b-specific cell selection. The protocol can be completed in 4 h, with an average yield of 450,000 and 145,000 viable cells per gram of white and grey matter tissue respectively. This method allows for the immediate phenotyping of microglia in relation to brain donor clinical variables, and shows the microglia population to be distinguishable from autologous choroid plexus macrophages. This protocol has been applied to samples from over 100 brain donors from the Netherlands Brain Bank, providing a robust dataset to analyze the effects of age, post-mortem delay, brain acidity, and neurological diagnosis on microglia yield and phenotype. Our data show that cerebrospinal fluid pH is positively correlated to microglial cell yield, but donor age and post-mortem delay do not negatively affect viable microglia yield. Analysis of CD45 and CD11b expression showed that changes in microglia phenotype can be attributed to a neurological diagnosis, and are not influenced by variation in ante- and post-mortem parameters. Cryogenic storage of primary microglia was shown to be possible, albeit with variable levels of recovery and effects on phenotype and RNA quality. Microglial gene expression substantially changed due to culture, including the loss of the microglia-specific markers, showing the importance of immediate microglia phenotyping. We conclude that primary microglia can be isolated effectively and rapidly from human post-mortem brain tissue, allowing for the study of the microglial population in light of the neuropathological status of the donor.

Glial fibrillary acidic protein (GFAP) is the characteristic intermediate filament (IF) protein in astrocytes. Expression of its main isoforms, GFAPα and GFAPδ, varies in astrocytes and astrocytoma implying a potential regulatory role in astrocyte physiology and pathology. An IF-network is a dynamic structure and has been functionally linked to cell motility, proliferation, and morphology. There is a constant exchange of IF-proteins with the network. To study differences in the dynamic properties of GFAPα and GFAPδ, we performed fluorescence recovery after photobleaching experiments on astrocytoma cells with fluorescently tagged GFAPs. Here, we show for the first time that the exchange of GFP–GFAPδ was significantly slower than the exchange of GFP–GFAPα with the IF-network. Furthermore, a collapsed IF-network, induced by GFAPδ expression, led to a further decrease in fluorescence recovery of both GFP–GFAPα and GFP–GFAPδ. This altered IF-network also changed cell morphology and the focal adhesion size, but did not alter cell migration or proliferation. Our study provides further insight into the modulation of the dynamic properties and functional consequences of the IF-network composition.

Multiple sclerosis is a serious neurological disorder, resulting in e.g., sensory, motor and cognitive deficits. A critical pathological aspect of multiple sclerosis (MS) is the influx of immunomodulatory cells into the central nervous system (CNS). Identification of key players that regulate cellular trafficking into the CNS may lead to the development of more selective treatment to halt this process. The multifunctional enzyme tissue Transglutaminase (TG2) can participate in various inflammation-related processes, and is known to be expressed in the CNS. In the present study, we question whether TG2 activity contributes to the pathogenesis of experimental MS, and could be a novel therapeutic target. In human post-mortem material, we showed the appearance of TG2 immunoreactivity in leukocytes in MS lesions, and particular in macrophages in rat chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE), an experimental MS model. Clinical deficits as observed in mouse EAE were reduced in TG2 knock-out mice compared to littermate wild-type mice, supporting a role of TG2 in EAE pathogenesis. To establish if the enzyme TG2 represents an attractive therapeutic target, cr-EAE rats were treated with TG2 activity inhibitors during ongoing disease. Reduction of TG2 activity in cr-EAE animals dramatically attenuated clinical deficits and demyelination. The mechanism underlying these beneficial effects pointed toward a reduction in macrophage migration into the CNS due to attenuated cytoskeletal flexibility and RhoA GTPase activity. Moreover, iNOS and TNFα levels were selectively reduced in the CNS of cr-EAE rats treated with a TG2 activity inhibitor, whereas other relevant inflammatory mediators were not affected in CNS or spleen by reducing TG2 activity. We conclude that modulating TG2 activity opens new avenues for therapeutic intervention in MS which does not affect peripheral levels of inflammatory mediators.

Glioblastoma multiforme (GBM) is the most common primary brain tumor and is without exception lethal. GBMs modify the immune system, which contributes to the aggressive nature of the disease. Particularly, cells of the monocytic lineage, including monocytes, macrophages and microglia, are affected. We investigated the influence of GBM‐derived extracellular vesicles (EVs) on the phenotype of monocytic cells. Proteomic profiling showed GBM EVs to be enriched with proteins functioning in extracellular matrix interaction and leukocyte migration. GBM EVs appeared to skew the differentiation of peripheral blood‐derived monocytes to alternatively activated/M2‐type macrophages. This was observed for EVs from an established cell line, as well as for EVs from primary cultures of GBM stem‐like cells (GSCs). Unlike EVs of non‐GBM origin, GBM EVs induced modified expression of cell surface proteins, modified cytokine secretion (e.g., an increase in vascular endothelial growth factor and IL‐6) and increased phagocytic capacity of the macrophages. Most pronounced effects were observed upon incubation with EVs from mesenchymal GSCs. GSC EVs also affected primary human microglia, resulting in increased expression of Membrane type 1‐matrix metalloproteinase, a marker for GBM microglia and functioning as tumor‐supportive factor. In conclusion, GBM‐derived EVs can modify cells of the monocytic lineage, which acquire characteristics that resemble the tumor‐supportive phenotypes observed in patients.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system, leading to severe neurological deficits. Current MS treatment regimens, consist of immunomodulatory agents aiming to reduce the rate of relapses. However, these agents are usually insufficient to treat chronic neurological disability. A promising perspective for future therapy of MS is the regeneration of lesions with replacement of the damaged oligodendrocytes or neurons. Therapies targeting to the enhancement of endogenous remyelination, aim to promote the activation of either the parenchymal oligodendrocyte progenitor cells or the subventricular zone-derived neural stem cells (NSCs). Less studied but highly potent, is the strategy of neuronal regeneration with endogenous NSCs that although being linked to numerous limitations, is anticipated to ameliorate cognitive disability in MS. Focusing on the forebrain, this review highlights the role of NSCs in the regeneration of MS lesions.

Neural progenitor cells (NPCs) in the subventricular zone (SVZ) hold promise for future therapy for neurodegenerative disorders, because the stimulation of adult neurogenesis could potentially restore the function of degenerating neurons and glia. To obtain more knowledge on these NPCs, we developed a method to specifically isolate NPCs from postmortem adult human brains based on the expression of the specific human adult neural stem/progenitor cell marker glial fibrillary acidic protein δ (GFAPδ). An extensive immunophenotyping analysis for cell surface markers resulted in the observation that CD271 was limited to the SVZ-derived GFAPδ-positive cells. CD271(+) cells developed into neurospheres and could be differentiated into astrocytes, neurons, and oligodendrocytes. We are the first to show that a pure population of NPCs can be isolated from the adult human SVZ, which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis.

Glial Fibrillary Acidic Protein (GFAP) is the main intermediate filament in astrocytes and is regulated by epigenetic mechanisms during development. We demonstrate that histone acetylation controls GFAP expression also in mature astrocytes. Inhibition of histone deacetylases (HDACs) with Trichostatin-A or Sodium-butyrate reduced GFAP expression in primary human astrocytes and astrocytoma cells. Since splicing occurs co-transcriptional, we investigated whether histone acetylation changes the ratio between the canonical isoform GFAPα and the alternative GFAPδ splice-variant. We observed that decreased transcription of GFAP enhanced alternative isoform expression, as HDAC inhibition increased the GFAPδ/α ratio favouring GFAPδ. Expression of GFAPδ was dependent on the presence and binding of the splicing factors of the SR protein family. Inhibition of HDAC activity also resulted in aggregation of the GFAP network, reminiscent to our earlier findings of a GFAPδ-induced network collapse. Together, our data demonstrate that HDAC inhibition results in changes in transcription, splicing, and organization of GFAP. These data imply that a tight regulation of histone acetylation in astrocytes is essential, since dysregulation of gene expression causes aggregation of GFAP, a hallmark of human diseases like Alexander's disease.

Astrocytes emerge as crucial cells for proper neuronal functioning in the developing and adult brain. Neurons and astrocytes are sequentially generated from the same pool of neural stem cells (NSCs). Tight regulation of the neuron-to-astrocyte switch is critical for (1) the generation of a balanced number of astrocytes and neurons and (2) neuronal circuit formation, since newborn astrocytes regulate synapse formation. This review focuses on signaling pathways that instruct astrogenesis, incorporating recently discovered intrinsic and extrinsic regulators. The canonical pathway of astrocytic gene expression, JAK/STAT signaling, is inhibited during neurogenesis to prevent premature astrocyte differentiation. At the onset of astrogenesis, Notch signaling induces epigenetic remodeling of astrocytic genes like glial fibrillary acidic protein to change NSC competence. In turn, astrogenesis is initiated by signals received from newborn neurons. We highlight how key molecular pathways like JAK/STAT and Notch are integrated in a complex network of environmental signals and epigenetic and transcriptional regulators to determine NSC differentiation. It is essential to understand NSC differentiation in respect to future NSC-based therapies for brain diseases, as transplanted NSCs preferentially become astrocytes. As emphasized in this review, many clues in this respect can be learned from development.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

The basis of gender differences in the prevalence and clinical progression of multiple sclerosis (MS) is not understood. Here, we identify gender-specific responses in steroid synthesis and signaling in the brains of MS patients as possible contributors to these differences. We investigated gene expression changes in these pathways and of inflammatory cytokines in MS lesions and normal-appearing white matter (NAWM) of male and female patients (n=21) and control NAWM (n=14) using quantitative polymerase chain reaction (25 MS lesions, 21 MS NAWM, and 14 control NAWM) and immunohistochemistry (3-4 sections per group). In MS lesions in males, there was local upregulation of aromatase (an enzyme involved in estrogen biosynthesis), estrogen receptor-β (ERβ), and tumor necrosis factor (TNF) mRNA; whereas in females, there was local upregulation of 3β-hydroxysteroid-dehydrogenase, a progesterone synthetic enzyme, and of progesterone receptor. Astrocytes in the rim and center of MS lesions were found to be the primary source of steroidogenic enzyme and receptor expression. Aromatase and ERα mRNA levels were positively correlated with that of TNF in primary cultures of human microglia and astrocytes; TNF caused increased ERα, suggesting that inflammatory signals stimulate estrogen signaling in this cell type. Together, these findings suggest that there are gender differences in the CNS of MS patients that may affect lesion pathogenesis, that is, in males, estrogen synthesis and signaling are induced; whereas in females, progestogen synthesis and signaling are induced. These differences may represent contributing factors to gender differences in the prevalence and course of MS.

Glial fibrillary acidic protein (GFAP) is the characteristic protein in the intermediate filament (IF) network of astrocytes. At least 10 different GFAP splice isoforms are known to be expressed, and the main isoforms are GFAPα and GFAPδ. The canonical GFAPα isoform is able to assemble into filaments by itself. GFAPδ, however, is assembly compromised, but low amounts of GFAPδ ( ~ 10% of total GFAP) are tolerated into an IF network composed of vimentin or GFAPα. An increasing amount of GFAPδ will lead to an imbalance with the other IFs present in the cells, and will cause a collapse of the IF network. An IF network is a dynamic structure and IF proteins continuously assemble and disassemble into the network. To study potential differences in the dynamic properties of GFAPα and GFAPδ, both in a physiological and a collapsed IF network, we performed a fluorescence recovery after photobleaching (FRAP) analysis on astrocytoma cells transfected with fluorescently-tagged GFAPs. Here we show for the first time that exchange of GFP-GFAPδ (t1/2 = 2.3 min) into the IF network of astrocytoma cells was significantly slower than the exchange of GFP-GFAPα (t1/2 = 1.1 min). Furthermore, we provide preliminary data that a collapsed IF network, induced by a high GFAPδ expression, led to an even more pronounced decrease in fluorescent recovery of both GFP-GFAPα (t1/2 = 4.5 min) and GFP-GFAPδ (t1/2 = 5.5 min). In the collapsed condition, however, the difference in dynamic properties between GFPGFAPα and GFP-GFAPδ was not present anymore. Our study provides a first insight into biochemical properties of two GFAP isoforms that leads to differences in IF network dynamics. As the IF cytoskeleton has been implicated in cell signaling, a change in the dynamic properties of the network could play a role in regulating cellular processes. More importantly, a GFAP collapse mimicks GFAP aggregates present in Rosenthal fibers in Alexander Disease and some glioma subyptes, therefore our data might help to understand the functional consequences of these aggregates in astrocytes.

Kinesin family member 21b (kif21b) is one of the few multiple sclerosis (MS) risk genes with a presumed central nervous system function. Kif21b belongs to the kinesin family, proteins involved in intracellular transport of proteins and organelles. We hypothesised that kif21b is involved in the neurodegenerative component of MS and Alzheimer's (AD) disease. Post-mortem kinesin expression was assessed in 50 MS, 58 age and gender matched non-demented controls (NDC) and 50 AD. Kif21b expression was five-fold increased in AD compared to MS and NDC aged below 62 years (p = 8*10(-5)), three-fold between 62-72 years (p = 0.005) and not different above 72 years. No significant differences were observed between MS and NDC. In AD, kif21b expression was two-fold increased in Braak stage 6 (scoring for density of neurofibrillary tangles) compared with stage 5 (p = 0.003). In MS patients, kif21b correlated with the extent of grey matter demyelination (Spearman's rho = 0.31, p = 0.03). Abundant kif21b, defined as expression above the median, was associated with a two-fold accelerated development of the Kurtzke Expanded Disability Status Scale (EDSS) 6.0 (median time in low kif21b group 16 years vs. high kif21b 7.5 years, log-rank test p = 0.04) in MS. Given the genetic association of kif21b with MS, the results were stratified according to rs12122721[A] single nucleotide polymorphism (SNP). No association was found between kif21b expression or the time to EDSS 6 in kif21b risk SNP carriers compared to non-risk carriers. Kif21b was expressed in astrocytes in addition to neurons. Upon astrocyte activation, kif21b increased nine-fold. Abundant kif21b expression is associated with severe MS and AD pathology and with accelerated neurodegeneration independent of the kif21b risk SNP.

Remyelination following central nervous system demyelination is essential to prevent axon degeneration. However, remyelination ultimately fails in demyelinating diseases such as multiple sclerosis. This failure of remyelination is likely mediated by many factors, including changes in the extracellular signalling environment. Here, we examined the expression of the extracellular matrix molecule fibronectin on demyelinating injury and how this affects remyelination by oligodendrocytes progenitors. In toxin-induced lesions undergoing efficient remyelination, fibronectin expression was transiently increased within demyelinated areas and declined as remyelination proceeded. Fibronectin levels increased both by leakage from the blood circulation and by production from central nervous system resident cells. In chronically demyelinated multiple sclerosis lesions, fibronectin expression persisted in the form of aggregates, which may render fibronectin resistant to degradation. Aggregation of fibronectin was similarly observed at the relapse phase of chronic experimental autoimmune encephalitis, but not on toxin-induced demyelination, suggesting that fibronectin aggregation is mediated by inflammation-induced demyelination. Indeed, the inflammatory mediator lipopolysaccharide induced fibronectin aggregation by astrocytes. Most intriguingly, injection of astrocyte-derived fibronectin aggregates in toxin-induced demyelinated lesions inhibited oligodendrocyte differentiation and remyelination, and fibronectin aggregates are barely expressed in remyelinated multiple sclerosis lesions. Therefore, these findings suggest that fibronectin aggregates within multiple sclerosis lesions contribute to remyelination failure. Hence, the inhibitory signals induced by fibronectin aggregates or factors that affect fibronectin aggregation could be potential therapeutic targets for promoting remyelination.