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.
The success of clinical trials of selective B cell depletion in patients with relapsing multiple sclerosis (MS) indicates B cells are important contributors to peripheral immune responses involved in ...the development of new relapses. Such B cell contribution to peripheral inflammation likely involves antibody-independent mechanisms. Of growing interest is the potential that B cells, within the MS central nervous system (CNS), may also contribute to the propagation of CNS-compartmentalized inflammation in progressive (non-relapsing) disease. B cells are known to persist in the inflamed MS CNS and are more recently described as concentrated in meningeal immune-cell aggregates, adjacent to the subpial cortical injury which has been associated with progressive disease. How B cells are fostered within the MS CNS and how they may contribute locally to the propagation of CNS-compartmentalized inflammation remain to be elucidated.
We considered whether activated human astrocytes might contribute to B cell survival and function through soluble factors. B cells from healthy controls (HC) and untreated MS patients were exposed to primary human astrocytes that were either maintained under basal culture conditions (non-activated) or pre-activated with standard inflammatory signals. B cell exposure to astrocytes included direct co-culture, co-culture in transwells, or exposure to astrocyte-conditioned medium. Following the different exposures, B cell survival and expression of T cell co-stimulatory molecules were assessed by flow cytometry, as was the ability of differentially exposed B cells to induce activation of allogeneic T cells.
Secreted factors from both non-activated and activated human astrocytes robustly supported human B cell survival. Soluble products of pre-activated astrocytes also induced B cell upregulation of antigen-presenting cell machinery, and these B cells, in turn, were more efficient activators of T cells. Astrocyte-soluble factors could support survival and activation of B cell subsets implicated in MS, including memory B cells from patients with both relapsing and progressive forms of disease.
Our findings point to a potential mechanism whereby activated astrocytes in the inflamed MS CNS not only promote a B cell fostering environment, but also actively support the ability of B cells to contribute to the propagation of CNS-compartmentalized inflammation, now thought to play key roles in progressive disease.
Early multiple sclerosis lesions feature relative preservation of oligodendrocyte cell bodies with dying back retraction of their myelinating processes. Cell loss occurs with disease progression. ...Putative injury mediators include metabolic stress (low glucose/nutrient), pro-inflammatory mediators (interferon γ and tumour necrosis factor α), and excitotoxins (glutamate). Our objective was to compare the impact of these disease relevant mediators on the injury responses of human mature oligodendrocytes. In the current study, we determined the effects of these mediators on process extension and survival of human brain derived mature oligodendrocytes in vitro and used bulk RNA sequencing to identify distinct effector mechanisms that underlie the responses. All mediators induced significant process retraction of the oligodendrocytes in dissociated cell culture. Only metabolic stress (low glucose/nutrient) conditions resulted in delayed (4-6 days) non-apoptotic cell death. Metabolic effects were associated with induction of the integrated stress response, which can be protective or contribute to cell injury dependent on its level and duration of activation. Addition of Sephin1, an agonist of the integrated stress response induced process retraction under control conditions and further enhanced retraction under metabolic stress conditions. The antagonist ISRIB restored process outgrowth under stress conditions, and if added to already stressed cells, reduced delayed cell death and prolonged the period in which recovery could occur. Inflammatory cytokine functional effects were associated with activation of multiple signalling pathways (including Jak/Stat-1) that regulate process outgrowth, without integrated stress response induction. Glutamate application produced limited transcriptional changes suggesting a contribution of effects directly on cell processes. Our comparative studies indicate the need to consider both the specific injury mediators and the distinct cellular mechanisms of responses to them by human oligodendrocytes to identify effective neuroprotective therapies for multiple sclerosis.
Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to ...repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.
•14-3-3 adaptor proteins facilitate axon growth•Stabilization of 14-3-3 complexes with Fusicoccin-A stimulates axon regeneration•Fusicoccin-A targets a complex between 14-3-3 and the stress response regulator GCN1•GCN1 acts as an intrinsic brake on neurite outgrowth
Kaplan et al. describe a novel pharmacological strategy with a unique mechanism of action to enhance axon regeneration in the injured CNS. Fusicoccin-A stabilizes 14-3-3 protein complexes and stimulates nerve regeneration, in part through modulation of the stress response regulator GCN1.
The contribution of local factors to the activation of immune cells infiltrating the CNS of patients with multiple sclerosis (MS) remains to be defined. The cytokine IL-15 is pivotal in the ...maintenance and activation of CD8 T lymphocytes, a prominent lymphocyte population found in MS lesions. We investigated whether astrocytes are a functional source of IL-15 sufficient to enhance CD8 T lymphocyte responses and whether they provide IL-15 in the inflamed CNS of patients with MS. We observed that human astrocytes in primary cultures increased surface IL-15 levels upon activation with combinations of proinflammatory cytokines. Expanded human myelin autoreactive CD8 T lymphocytes cultured with such activated astrocytes displayed elevated lytic enzyme content, NKG2D expression, and Ag-specific cytotoxicity. These functional enhancements were abrogated by anti-IL-15-blocking Abs. Immunohistochemical analysis of brain tissue sections obtained from patients with MS demonstrated colocalization for IL-15 and the astrocyte marker glial fibrillary acidic protein within white matter lesions. The majority of astrocytes (80-90%) present in demyelinating MS lesions expressed IL-15, whereas few astrocytes in normal control brain sections had detectable IL-15. IL-15 could be detected in the majority of Iba-1-expressing microglia in the control sections, albeit in lower numbers when compared with microglia/macrophages in MS lesions. Furthermore, infiltrating CD8 T lymphocytes in MS lesions were in close proximity to IL-15-expressing cells. Astrocyte production of IL-15 resulting in the activation of CD8 T lymphocytes ascribes a role for these cells as contributors to the exacerbation of tissue damage during MS pathogenesis.
In multiple sclerosis, cerebellar symptoms are associated with clinical impairment and an increased likelihood of progressive course. Cortical atrophy and synaptic dysfunction play a prominent role ...in cerebellar pathology and although the dentate nucleus is a predilection site for lesion development, structural synaptic changes in this region remain largely unexplored. Moreover, the mechanisms leading to synaptic dysfunction have not yet been investigated at an ultrastructural level in multiple sclerosis. Here, we report on synaptic changes of dentate nuclei in post‐mortem cerebella of 16 multiple sclerosis patients and eight controls at the histological level as well as an electron microscopy evaluation of afferent synapses of the cerebellar dentate and pontine nuclei of one multiple sclerosis patient and one control. We found a significant reduction of afferent dentate synapses in multiple sclerosis, irrespective of the presence of demyelination, and a close relationship between glial processes and dentate synapses. Ultrastructurally, we show autophagosomes containing degradation products of synaptic vesicles within dendrites, residual bodies within intact‐appearing axons and free postsynaptic densities opposed to astrocytic appendages. Our study demonstrates loss of dentate afferent synapses and provides, for the first time, ultrastructural evidence pointing towards neuron‐autonomous and neuroglia‐mediated mechanisms of synaptic degradation in chronic multiple sclerosis.
Neuroimmune interaction during development is strongly implicated in the pathogenesis of neurodevelopmental disorders, but the mechanisms that cause neuronal circuit dysregulation are not well ...understood. We performed
imaging of the developing retinotectal system in the larval zebrafish to characterize the effects of immune system activation on refinement of an archetypal sensory processing circuit. Acute inflammatory insult induced hyperdynamic remodeling of developing retinal axons in larval fish and increased axon arbor elaboration over days. Using calcium imaging in GCaMP6s transgenic fish, we showed that these morphologic changes were accompanied by a shift toward decreased visual acuity in tectal cells. This finding was supported by poorer performance in a visually guided behavioral task. We further found that the pro-inflammatory cytokine, interleukin-1β (IL-1β), is upregulated by the inflammatory insult, and that downregulation of IL-1β abrogated the effects of inflammation on axonal dynamics and growth. Moreover, baseline branching of the retinal ganglion cell arbors in IL-1β morphant animals was significantly different from that in control larvae, and their performance in a predation assay was impaired, indicating a role for this cytokine in normal neuronal development. This work establishes a simple and powerful non-mammalian model of developmental immune activation and demonstrates a role for IL-1β in mediating the pathologic effects of inflammation on neuronal circuit development.
Maternal immune activation can increase the risk of neurodevelopmental disorders in offspring; however, the mechanisms involved are not fully understood. Using a non-mammalian vertebrate model of developmental immune activation, we show that even brief activation of inflammatory pathways has immediate and long-term effects on the arborization of axons, and that these morphologic changes have functional and behavioral consequences. Finally, we show that the pro-inflammatory cytokine IL-1β plays an essential role in both the effects of inflammation on circuit formation and normal axonal development. Our data add to a growing body of evidence supporting epidemiological studies linking immune activation to neurodevelopmental disorders, and help shed light on the molecular and cellular processes that contribute to the etiology of these disorders.
ABSTRACT
Fingolimod (FTY720) is a sphingosine 1‐phosphate (S1P) receptor modulator that regulates lymphocyte trafficking and exerts pleiotropic actions on oligodendrocytes (OLGs) and other neural ...cells. The purpose of this study was to investigate the role of S1P receptors in a non‐T‐cell model of demyelination, the cuprizone (cupr) model in C57BL/6 mice. Treatment with FTY720 (1 mg/kg) led to attenuated injury to OLGs, myelin, and axons in the corpus callosum (percentage of myelinated fibers was 44.7% in cupr‐water and 63% in cupr‐FTY720). Reactive astrogliosis and microgliosis were ameliorated when FTY720 was given from d 1, but astrogliosis was augmented when FTY720 was given from wk 4–9. FTY720 did not promote remyelination in this model. The protective effect of FTY720 was associated with decreased interleukin‐1 β and CCL2 transcripts in the corpus callosum, as well as altered S1P1 expression. Targeted deletion of S1P1 in OLG lineage cells did not lead to obvious clinical phenotype, but resulted in subtle abnormalities in myelin and an increased susceptibility to cupr‐induced demyelination. We conclude that S1P receptors expressed by neuroglia are involved in regulating the response to injury, and CNS effects of FTY720 could contribute to its favorable therapeutic response in multiple sclerosis.—Kim, H. J., Miron, V. E., Dukala, D., Proia, R. L., Ludwin, S. K., Traka, M., Antel, J. P., Soliven, B. Neurobiological effects of sphingosine 1‐phosphate receptor modulation in the cuprizone model. FASEB J. 25, 1509–1518 (2011). www.fasebj.org
Myelin, the membrane surrounding neuronal axons, is critical for central nervous system (CNS) function. Injury to myelin-forming oligodendrocytes (OL) in chronic neurological diseases (e.g. multiple ...sclerosis) ranges from sublethal to lethal, leading to OL dysfunction and myelin pathology, and consequent deleterious impacts on axonal health that drive clinical impairments. This is regulated by intrinsic factors such as heterogeneity and age, and extrinsic cellular and molecular interactions. Here, we discuss the responses of OLs to injury, and perspectives for therapeutic targeting. We put forward that targeting mature OL health in neurological disease is a promising therapeutic strategy to support CNS function.
The mechanisms whereby immune cells infiltrating the CNS in multiple sclerosis patients contribute to tissue injury remain to be defined. CD4 T cells are key players of this inflammatory response. ...Myelin-specific CD4 T cells expressing CD56, a surrogate marker of NK cells, were shown to be cytotoxic to human oligodendrocytes. Our aim was to identify NK-associated molecules expressed by human CD4 T cells that confer this oligodendrocyte-directed cytotoxicity. We observed that myelin-reactive CD4 T cell lines, as well as short-term PHA-activated CD4 T cells, can express NKG2C, the activating receptor interacting with HLA-E, a nonclassical MHC class I molecule. These cells coexpress CD56 and NKG2D, have elevated levels of cytotoxic molecules FasL, granzyme B, and perforin compared with their NKG2C-negative counterparts, and mediate significant in vitro cytotoxicity toward human oligodendrocytes, which upregulated HLA-E upon inflammatory cytokine treatment. A significantly elevated proportion of ex vivo peripheral blood CD4 T cells, but not CD8 T cells or NK cells, from multiple sclerosis patients express NKG2C compared with controls. In addition, immunohistochemical analyses showed that multiple sclerosis brain tissues display HLA-E(+) oligodendrocytes and NKG2C(+) CD4 T cells. Our results implicate a novel mechanism through which infiltrating CD4 T cells contribute to tissue injury in multiple sclerosis.
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