Promoting Central Nervous System Repair Research Articles

Microglial TNFR2 signaling regulates the inflammatory response after CNS injury in a sex-specific fashion

Microglia, the resident immune cells of the central nervous system (CNS), play a major role in damage progression and tissue remodeling after acute CNS injury, including ischemic stroke (IS) and spinal cord injury (SCI). Understanding the molecular mechanisms regulating microglial responses to injury may thus reveal novel therapeutic targets to promote CNS repair. Here, we investigated the role of microglial tumor necrosis factor receptor 2 (TNFR2), a transmembrane receptor previously associated with pro-survival and neuroprotective responses, in shaping the neuroinflammatory environment after CNS injury. By inducing experimental IS and SCI in Cx3cr1CreER:Tnfrsf1bfl/fl mice, selectively lacking TNFR2 in microglia, and corresponding Tnfrsf1bfl/fl littermate controls, we found that ablation of microglial TNFR2 significantly reduces lesion size and pro-inflammatory cytokine levels, and favors infiltration of leukocytes after injury. Interestingly, these effects were paralleled by opposite sex-specific modifications of microglial reactivity, which was found to be limited in female TNFR2-ablated mice compared to controls, whereas it was enhanced in males. In addition, we show that TNFR2 protein levels in the cerebrospinal fluid (CSF) of human subjects affected by IS and SCI, as well as healthy donors, significantly correlate with disease stage and severity, representing a valuable tool to monitor the inflammatory response after acute CNS injury. Hence, these results advance our understanding of the mechanisms regulating microglia reactivity after acute CNS injury, aiding the development of sex- and microglia-specific, personalized neuroregenerative strategies.

Brain region-specific enhancement of remyelination and prevention of demyelination by the CSF1R kinase inhibitor BLZ945

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system (CNS). While multiple effective immunomodulatory therapies for MS exist today, they lack the scope of promoting CNS repair, in particular remyelination. Microglia play a pivotal role in regulating myelination processes, and the colony-stimulating factor 1 (CSF-1) pathway is a key regulator for microglia differentiation and survival. Here, we investigated the effects of the CSF-1 receptor kinase inhibitor, BLZ945, on central myelination processes in the 5-week murine cuprizone model by non-invasive and longitudinal magnetic resonance imaging (MRI) and histology. Therapeutic 2-week BLZ945 treatment caused a brain region-specific enhancement of remyelination in the striatum/cortex, which was absent in the corpus callosum/external capsule. This beneficial effect correlated positively with microglia reduction, increased oligodendrocytes and astrogliosis. Prophylactic BLZ945 treatment prevented excessive demyelination in the corpus callosum by reducing microglia and increasing oligondendrocytes. In the external capsule oligodendrocytes were depleted but not microglia and a buildup of myelin debris and axonal damage was observed. A similar microglial dysfunction in the external capsule with an increase of myelin debris was obvious in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice treated with cuprizone. Finally, therapeutic BLZ945 treatment did not change the disease course in experimental autoimmune encephalomyelitis mice, a peripherally driven neuroinflammation model. Taken together, our data suggest that a short-term therapeutic inhibition of the CSF-1 receptor pathway by BLZ945 in the murine cuprizone model enhances central remyelination by modulating neuroinflammation. Thus, microglia-modulating therapies could be considered clinically for promoting myelination in combination with standard-of-care treatments in MS patients.

Recent Advances in the Study of Bipolar/Rod-Shaped Microglia and their Roles in Neurodegeneration.

Microglia are the resident immune cells of the central nervous system (CNS) and they contribute to primary inflammatory responses following CNS injuries. The morphology of microglia is closely associated with their functional activities. Most previous research efforts have attempted to delineate the role of ramified and amoeboid microglia in the pathogenesis of neurodegenerative diseases. In addition to ramified and amoeboid microglia, bipolar/rod-shaped microglia were first described by Franz Nissl in 1899 and their presence in the brain was closely associated with the pathology of infectious diseases and sleeping disorders. However, studies relating to bipolar/rod-shaped microglia are very limited, largely due to the lack of appropriate in vitro and in vivo experimental models. Recent studies have reported the formation of bipolar/rod-shaped microglia trains in in vivo models of CNS injury, including diffuse brain injury, focal transient ischemia, optic nerve transection and laser-induced ocular hypertension (OHT). These bipolar/rod-shaped microglia formed end-to-end alignments in close proximity to the adjacent injured axons, but they showed no interactions with blood vessels or other types of glial cell. Recent studies have also reported on a highly reproducible in vitro culture model system to enrich bipolar/rod-shaped microglia that acts as a powerful tool with which to characterize this form of microglia. The molecular aspects of bipolar/rod-shaped microglia are of great interest in the field of CNS repair. This review article focuses on studies relating to the morphology and transformation of microglia into the bipolar/rod-shaped form, along with the differential gene expression and spatial distribution of bipolar/rod-shaped microglia in normal and pathological CNSs. The spatial arrangement of bipolar/rod-shaped microglia is crucial in the reorganization and remodeling of neuronal and synaptic circuitry following CNS injuries. Finally, we discuss the potential neuroprotective roles of bipolar/rod-shaped microglia, and the possibility of transforming ramified/amoeboid microglia into bipolar/rod-shaped microglia. This will be of considerable clinical benefit in the development of novel therapeutic strategies for treating various neurodegenerative diseases and promoting CNS repair after injury.

Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki.

Neuron glia antigen 2 (NG2)-positive glia are repair cells that proliferate upon central nervous system (CNS) damage, promoting functional recovery. However, repair is limited because of the failure of the newly produced glial cells to differentiate. It is a key goal to discover how to regulate NG2 to enable glial proliferation and differentiation conducive to repair. Drosophila has an NG2 homologue called kon-tiki (kon), of unknown CNS function. We show that kon promotes repair and identify the underlying mechanism. Crush injury up-regulates kon expression downstream of Notch. Kon in turn induces glial proliferation and initiates glial differentiation by activating glial genes and prospero (pros). Two negative feedback loops with Notch and Pros allow Kon to drive the homeostatic regulation required for repair. By modulating Kon levels in glia, we could prevent or promote CNS repair. Thus, the functional links between Kon, Notch, and Pros are essential for, and can drive, repair. Analogous mechanisms could promote CNS repair in mammals.

Role of MicroRNAs in Development of Immune Cells and Nervous System and their Relation to Multiple Sclerosis

Introduction: MicroRNAs (miRNAs) are small and non-coding ribonucleic acids that play critical roles in regulation of host genome expression at post-transcriptional level. An individual miRNA is able to down-regulate multiple targeted mRNA transcripts. Therefore, minor changes in a miRNA expression may lead to significant alterations in the expression of different genes. During last two decades, miRNAs have emerged as key regulators of immune cell lineage differentiation, maturation, maintenance of immune homeostasis, and normal function. Multiples sclerosis (MS) is a chronic inflammatory disease that is characterized by infiltration of lymphocytes into the central nervous system (CNS), demyelination and axonal degeneration. Although causes of MS are still unknown, it is widely accepted that novel drug targets need to focus on both decreasing inflammation and promoting CNS repair. Recent researches about MS disease have shown that miRNAs are dysregulated in the immune system and CNS, which shows their role in the MS pathogenesis. Conclusion: Identification of specific expression patterns of miRNA in autoimmune diseases and a further comprehensive understanding of their role in the pathogenesis of different diseases offers promise of not only novel molecular diagnostic markers but also new gene therapy strategies for treating inflammatory autoimmune diseases. In this study, we review the latest findings about miRNA biogenesis and signatures in the CNS and immune cells of MS patients.

Hyaluronan Synthesis, Catabolism, and Signaling in Neurodegenerative Diseases.

The glycosaminoglycan hyaluronan (HA), a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS). HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair.

Structure of the LINGO-1-anti-LINGO-1 Li81 antibody complex provides insights into the biology of LINGO-1 and the mechanism of action of the antibody therapy.

Multiple sclerosis (MS) is an autoimmune-inflammatory disease of the central nervous system (CNS) with prominent demyelination and axonal injury. While most MS therapies target the immunologic response, there is a large unmet need for treatments that can promote CNS repair. LINGO-1 (leucine-rich repeat and Ig-containing Nogo receptor interacting protein-1) is a membrane protein selectively expressed in the CNS that suppresses myelination, preventing the repair of damaged axons. We are investigating LINGO-1 antagonist antibodies that lead to remyelination as a new paradigm for treatment of individuals with MS. The anti-LINGO-1 Li81 antibody,BIIB033, is currently in clinical trials and is the first MS treatment targeting CNS repair. Here, to elucidate the mechanism of action of the antibody, we solved the crystal structure of the LINGO-1-Li81 Fab complex and used biochemical and functional studies to investigate structure-function relationships. Li81 binds to the convex surface of the leucine-rich repeat domain of LINGO-1 within repeats 4-8. Fab binding blocks contact points used in the oligomerization of LINGO-1 and produces a stable complex containing two copies each of LINGO-1 and Fab that results from a rearrangement of contacts stabilizing the quaternary structure of LINGO-1. The formation of the LINGO-1-Li81 Fab complex masks functional epitopes within the Ig domain of LINGO-1 that are important for its biologic activity in oligodendrocyte differentiation. These studies provide new insights into the structure and biology of LINGO-1 and how Li81 monoclonal antibody can block its function.

MicroRNA dysregulation in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by central nervous system (CNS) demyelination and axonal degeneration. Although the cause of MS is still unknown, it is widely accepted that novel drug targets need to focus on both decreasing inflammation and promoting CNS repair. In MS and experimental autoimmune encephalomyelitis, non-coding small microRNAs (miRNAs) are dysregulated in the immune system and CNS. Since individual miRNAs are able to down-regulate multiple targeted mRNA transcripts, even minor changes in miRNA expression may lead to significant alterations in gene expression. Herein, we review miRNA signatures reported in CNS tissue and immune cells of MS patients and consider how altered miRNA expression may influence MS pathology.

Mechanism of action of somatic stem cell treatments: towards the concept of therapeutic plasticity

The initial perception that transplantation of stem cells could serve only to replace damaged cells has continued to be modifi ed as evidence for a protective multifaceted function of transplanted cells has emerged. Neural stem/precursor cells (NPC) were the fi rst to be shown to use this strategy to protect the central nervous system (CNS) from infl ammatory insults (1 – 5). Subsequently similar functions by other types of somatic stem cells (e.g. mesenchymal stromal cells; MSC) have been described (6). Currently the ability of transplanted stem cells to protect tissues from diverse injury using multifaceted ‘ bystander strategies ’ has been substantiated in several experiments. Somatic stem cells promote CNS repair following intralesional as well as systemic injection by producing in situ a milieu of protective molecules whose release is regulated, both temporally and spatially, by environmental demands. The milieu contains immunomodulatory substances, trophic growth factors, stem cell regulators, angiogenic factors, scavengers, etc.: all molecules constitutively expressed by somatic stem cells for maintaining tissue homeostasis during development and in adult life. The concept of ‘ therapeutic platicity ’ that is now emerging (7) also reconciles data showing that stem cells other than NPC, although endowed with negligible transdifferentiation capability, may have a benefi cial role in CNS repair. In four articles of this issue of Cytotherapy the concept of therapeutic plasticity of somatic stem cells is presented further. Although these reports do not completely clarify the mechanisms underlying this therapeutic plasticity, they demonstrate that transplantation of various somatic stem cells via different routes ameliorates diverse neurologic diseases without an overt terminal differentiation and functional integration of the transplanted cells. Alexanian et al. (8) observe that delayed intralesional transplantation of human glial-restricted NPC in rats affected by thoracic spinal cord injury ameliorates sensorimotor defi cits. He et al. (9) report that bone marrow (BM)-derived MSC and endothelial progenitor cells transplanted intravenously (i.v.) protect rats from focal ischemia via the secretion of different combinations of growth factors. Interestingly, an additive benefi cial effect was observed when the two different cell sources were co-transplanted. Li et al. (10) focus on a mouse model of muscular dystrophy where mean survival as well as loss of dystrophin and utrophin expression is delayed by i.v. transplantation of rat-derived MSC. Attar et al. (11) describe preliminary results of intralesional transplantation of BM-derived hematopoietic stem cells along with BM-derived mononuclear cells in four patients with spinal cord injury. Although the possibility of using bystander strategies as protection from tissue injury appears to be a very promising therapeutic use of somatic stem cells, we still need to resolve unsolved key questions before clinical translation. One crucial question is whether to transplant the cells heterotopically or homotopically. Transplantation is not without risks: co-transplantation of NPC and pancreatic islet cells under the kidney capsule of diabetic mice induced the generation, at the site of cell transplantation, of insulin-induced neuroblastoma-like neoplasms (12). Intracardiac transplantation of stem cell factor-overexpressing MSC into mice with myocardial infarction promoted the formation of a chest wall-invading soft tissue sarcoma (13). The unregulated transplantation Cytotherapy, 2011; 13: 6–7

Diffusible, membrane-bound, and extracellular matrix factors from olfactory ensheathing cells have different effects on the self-renewing and differentiating properties of neural stem cells

Transplantation of olfactory ensheathing cells (OECs) has been a promising strategy in enhancing central nervous system (CNS) regeneration. However, little is known about the effects of transplanted OECs on the self-renewal, neurogenesis, and oligodendrogenesis of neural stem cells (NSCs), which are known to play a very important role in the repair of damaged CNS tissue. In this study, we investigated the influence of diffusible, membrane-bound, and extracellular matrix factors from OECs on the self-renewal and differentiation properties of NSCs. We found that diffusible factors from cultured OECs promoted self-renewal, whereas the extracellular matrix molecules from OECs increased neurogenesis and oligodendrogenesis of NSCs. Furthermore, we demonstrated that directly coculturing OECs and NSCs inhibited not only self-renewal but also neurogenesis and oligodendrogenesis of NSCs. We propose three models for the interaction between transplanted OECs and endogenous NSCs. Our findings provide new insight into the ability of OECs to promote CNS repair and also indicate potential targets for manipulation of these cells to enhance their restorative ability.

A Neutralizing Anti-Nogo66 Receptor Monoclonal Antibody Reverses Inhibition of Neurite Outgrowth by Central Nervous System Myelin

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.

Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats

Contusive spinal cord injury (SCI) produces large fluid-, debris- and inflammatory cell-filled cystic cavities that lack structure to support significant axonal regeneration. The recent discovery of stem cells capable of generating central nervous system (CNS) tissues, coupled with success in neurotransplantation strategies, has renewed hope that repair and recovery from CNS trauma is possible. Based on results from several studies using bone marrow stromal cells (MSCs) to promote CNS repair, we transplanted MSCs into the rat SCI lesion cavity to further investigate their effects on functional recovery, lesion morphology, and axonal growth. We found that transplanted MSCs induced hindlimb airstepping—a spontaneous locomotor movement associated with activation of the stepping control circuitry—but did not alter the time course or extent of overground locomotor recovery. Using stereological techniques to describe spinal cord anatomy, we show that MSC transplants occupied the lesion cavity and were associated with preservation of host tissue and white matter (myelin), demonstrating that these cells exert neuroprotective effects. The tissue matrix formed by MSC grafts supported greater axonal growth than that found in specimens without grafts. Moreover, uniform random sampling of axon profiles revealed that the majority of neurites in MSC grafts were oriented with their long axis parallel to that of the spinal cord, suggesting longitudinally directed growth. Together, these studies support further investigation of marrow stromal cells as a potential SCI repair strategy.